The Inhibitory Activity of Citral against Malassezia furfur

Yi-Tsz Liu; Meng-Hwan Lee; Yin-Shen Lin; Wen-Lin Lai

doi:10.3390/pr10050802

The Inhibitory Activity of Citral against Malassezia furfur

Liu, Yi-Tsz;Lee, Meng-Hwan;Lin, Yin-Shen;Lai, Wen-Lin 2022-04-19 00:00:00 processes Article 1 2 2 1 , 3 , Yi-Tsz Liu , Meng-Hwan Lee , Yin-Shen Lin and Wen-Lin Lai * Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan; 1052039@mail.atri.org.tw Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute, Zhunan Township 35053, Taiwan; mhlee@mail.atri.org.tw (M.-H.L.); chris112783@mail.atri.org.tw (Y.-S.L.) Clinical Laboratory, Chung Shan Medical University Hospital, Taichung 40201, Taiwan * Correspondence: wllai@csmu.edu.tw; Tel.: +886-4-24730022 (ext. 12421) Abstract: The lipophilic yeast Malassezia furfur, is a member of the cutaneous commensal microbiota and is associated with several chronic diseases such as dandruff, pityriasis versicolor, folliculitis, and seborrheic dermatitis, that are often difﬁcult to treat with current therapies. The development of alternatively effective antifungal therapies is therefore of paramount importance. In this study, we investigated the treatment effect of citral on M. furfur. The minimal inhibitory concentration of citral for M. furfur was 200 g/mL, and the minimal fungicidal concentration was 300 g/mL. Citral signiﬁcantly increased the proportion of yeast cells to mycelial forms 2.6-fold. Phosphatidylserine externalization, DNA fragmentation, and metacaspase activation supported a citral-induced apop- tosis in M. furfur. Moreover, citral at sub-minimum inhibitory concentrations reduced the invasion of M. furfur in HaCaT keratinocytes. Finally, we demonstrated that citral inhibited IL-6 and TLR-2 expression and enhanced HBD-2 and TSLP expression in M. furfur-infected HaCaT keratinocytes. These results showed that citral has antifungal activity at high concentrations and can decrease the infection of M. furfur by modulating the keratinocyte immune responses at low concentrations. Our results suggest that citral is a potential candidate for topical therapeutic application for M. furfur-associated human skin diseases. Citation: Liu, Y.-T.; Lee, M.-H.; Lin, Keywords: Malassezia furfur; citral; yeast apoptosis; human keratinocyte; immunomodulation Y.-S.; Lai, W.-L. The Inhibitory Activity of Citral against Malassezia furfur. Processes 2022, 10, 802. https://doi.org/10.3390/pr10050802 1. Introduction Academic Editor: Maurizio Ventre The lipophilic yeast Malassezia furfur is a member of the cutaneous commensal micro- Received: 17 February 2022 biota of human skin, and is found particularly in areas rich in sebaceous gland content. Accepted: 17 April 2022 M. furfur infection can result in several chronic superﬁcial dermatitis such as dandruff, Published: 19 April 2022 pityriasis versicolor, seborrheic dermatitis, folliculitis, and atopic dermatitis. M. furfur is a dimorphic fungus that can alter its morphology from a unicellular yeast form to a Publisher’s Note: MDPI stays neutral mycelial form. M. furfur primarily shows a yeast form in normal conditions and standard with regard to jurisdictional claims in published maps and institutional afﬁl- cultures. However, it transforms into a mycelial form under some stimulations which play iations. a predominant role in the pathogenesis of some diseases, such as pityriasis versicolor [1–5]. Several azoles, such as ketoconazole (KTZ) in solutions, creams, gels, and shampoo forms are used in routine clinical treatment of M. furfur. However, KTZ demonstrates toxicity to mammalian cells and may cause urticarial [6]. M. furfur-related diseases are Copyright: © 2022 by the authors. often refractory to therapy and require extended use of antifungal and anti-inﬂammatory Licensee MDPI, Basel, Switzerland. medications, which may lead to drug resistance [7–9]. Therefore, ﬁnding a safe, effective, This article is an open access article and side-effect-free treatment is required. distributed under the terms and Plants and their derivatives are known sources of a variety of biologically active conditions of the Creative Commons components. They have great potential due to their low cost, low toxicity, and safety. Attribution (CC BY) license (https:// Previous studies have shown that extracts or puriﬁed ingredients from Trigonella foenum- creativecommons.org/licenses/by/ graecum, Asparagus racemosus, Hypericumper foratum, Dittrichia viscosa, and Vitis vinifera had 4.0/). Processes 2022, 10, 802. https://doi.org/10.3390/pr10050802 https://www.mdpi.com/journal/processes Processes 2022, 10, 802 2 of 13 treatment effects on Malassezia spp. infections [10–14]. Cymbopogon citratus, a herb and perennial tropical grass commonly known as lemon grass, is widely used in mid-tropical countries such as Southeast Asia, South America, and Africa as food seasoning and perfume material and as herbal medicines for its analgesic and anti-inﬂammatory properties [15]. Citral (3,7-dimethyl-2,6-octadienal) is the major constituent of Cymbopogon citratus and is classiﬁed as a “generally recognized as safe” (GRAS) substance and is used in food, perfume, and cosmetics and as a pharmaceutical component because of its lemon-like ﬂavor. Citral is a mixture of two isomeric acyclic aldehydes, geranial (trans-citral, citral A) and neral (cis-citral, citral B). Several studies have demonstrated that citral possesses antifungal, antimicrobial, and anti-inﬂammatory activities [16,17]. The antifungal activity exerted by citral has been demonstrated in varied conditions. Recently, it has been shown that citral can destroy the integrity of the cell membrane. Citral could also exert its antifungal effect by inhibiting ergosterol biosynthesis and mycelial growth. Citral inhibits fungal growth by damaging oxidative phosphorylation and cell membranes through massive ROS accumulation [12,16]. Citral could be added to many ﬁnished products. The maximum acceptable concentrations of citral in ﬁnished products are reviewed in the literature. For example, the maximum acceptable concentration of citral in hand-cream products is about 0.15%; in products applied to the hair with some hand contact is about 0.2%; and in products with body and hand exposure but which are primarily rinsed-off is about 1.2% [18]. Keratinocyte are a primary cell type in the epidermis that forms an essential barrier against invading microorganisms. Keratinocytes are also involved in innate immune de- fense mechanisms. Previous studies have shown that keratinocytes induces the production of pro-inﬂammatory cytokines by co-culturing with Malassezia yeasts [19,20]. Keratinocytes also produce different antimicrobial peptides, such as the defensins family, contributing to host defense against microorganisms. Cymbopogon citratus possesses various pharmacological activities, but little is known about the immunomodulating effects on keratinocytes. Although several studies have demonstrated that citral possesses antifungal, antimicrobial, and anti-inﬂammatory ac- tivities [16,17], no studies have investigated its antifungal activity and action mechanism against M. furfur. Citral’s yeast apoptotic process has been reported in common disease- causing yeasts such as Candida albican [3,21–23], but not in Malassezia spp. Therefore, this study aimed to investigate the inhibitory activity of citral against M. furfur and its im- munomodulatory effect on keratinocytes in vitro. 2. Materials and Methods 2.1. Materials Citral (Sigma-Aldrich, St. Louis, MO, USA) with 96% purity was dissolved in dimethyl sulfoxide (DMSO) to prepare the stock solution just before performing the assays. The antifungal drug KTZ (Sigma-Aldrich) stock solution was prepared using DMSO as the solvent and stored at 20 C. The ﬁnal concentration of DMSO in every assay was 1%. 2.2. Microorganism and Cultivation M. furfur BCRC 22,243 was purchased from the Bioresource Collection and Research Center (BCRC; Taiwan). To prepare budding yeast suspensions, the strain was grown in modiﬁed Dixon (mDixon) medium (3.6% malt extract, 2.0% desiccated ox bile, 0.6% peptone, 1.0% tween 40, and 0.2% oleic acid, pH 6.0) and incubated for three days at 30 C. For the human keratinocyte invasion experiment, M. furfur was cultured on agar plates of modiﬁed Leeming and Notman (mLNA) agar (1% peptone, 1% glucose, 0.2% yeast extract, 0.8% desiccated ox bile, 1% glycerol, 0.05% glycerol monostearate, 0.5% tween 60, 2% olive oil, and 1.5% agar, pH 6.0) for three days at 30 C [24]. 2.3. Antifungal Susceptibility Tests The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of citral to M. furfur were determined using the microdilution broth method. Serial Processes 2022, 10, 802 3 of 13 two-fold dilutions of citral were made in 24-well microtiter plates to obtain concentrations of 1.0 to 1000 g/mL. M. furfur was suspended in mDixon medium to the ﬁnal density of 5 10 CFU/mL. Each well was inoculated with 0.5 mL of the inoculum suspension and incubated at 30 C for 48 h. KTZ was utilized as the control drug. The MIC was deﬁned as the lowest drug concentration that would inhibit the visible growth of a microorganism after incubation. MFC was determined as the lowest drug concentration that killed >99.9% of the initial inoculum. To determine MFC, 50 L from each well showing no growth was spread on the mDixon plates and incubated at 30 C for 72 h [25]. In the time–killing assay, yeasts were treated with different concentrations of citral. After incubation at various time points, the cells were plated out on mDixon plates for viable counts. 2.4. Morphological Analysis M. furfur cells were stained with lactophenol cotton blue (LPCB) staining after being treated with different concentrations of citral for 48 h at 30 C. The proportions of yeast to mycelial conversion of M. furfur treated with various concentrations of citral were determined using a light microscopy at 1000 magniﬁcation [7]. 2.5. Analysis of Apoptosis Markers 2.5.1. Phosphatidylserine Externalization For early-stage apoptotic marker analysis, phosphatidylserine externalization was determined using the Annexin V-FITC apoptosis detection kit (BD Pharmingen, San Jose, CA, USA). M. furfur yeast cells (1 10 CFU/mL) were harvested by centrifugation, and washed and digested with lysing enzyme (20 mg/mL) and lyticase (50 U/L) in 0.1 M potassium phosphate buffer (PPB; pH 6.0) containing 1 M sorbitol for 2 h at 30 C. Protoplasts of M. furfur were incubated with citral or H O for 2 h at 30 C, washed and 2 2 resuspended in annexin binding buffer. Cells were incubated with 5 L/mL of annexin V-FITC and propidium iodide (PI) for 20 min. Flow cytometry was performed with a FACSCalibur ﬂow cytometer (Becton Dickinson, San Jose, CA, USA) [26]. 2.5.2. TUNEL Assay DNA fragmentation was analyzed by the terminal deoxynucleotidyl transferase dUTP TM nick-end labeling (TUNEL) assay using APO-DIRECT kit (BD Pharmingen) to observe late stage of apoptosis. Cells (1 10 CFU/mL) were treated with citral for 24 h at 30 C, then ﬁxed and protoplasted, labeled with DNA labeling solution, and stained with PI. Cells were then analyzed with a FACSCalibur ﬂow cytometer [27]. 2.5.3. Metacaspase Activation Activated metacaspases in M. furfur were measured using the CaspACE FITC-VAD- FMK In Situ Marker (Promega, Madison WI). Cells (1 10 CFU/mL) were treated with citral or H O for 3 h at 30 C. The cells were washed in PBS, suspended in a staining 2 2 solution containing 10 M FITC-VAD-FMK, incubated for 20 min at room temperature in the dark, and analyzed with a FACSCalibur ﬂow cytometer [28]. 2.6. Cell Culture A human keratinocyte (HaCaT) cell line was cultured as monolayers in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 g/mL streptomycin at 37 C with 5% CO . The medium was changed every 2 days. 2.7. MTT Assay The cytotoxic effect of citral on HaCaT cells was determined using MTT assay. HaCaT cells (5 10 cells/mL) were treated with 25–800 g/mL citral at 37 C for 48 and 72 h. The medium was removed and the cells were washed once with PBS; DMEM containing Processes 2022, 10, 802 4 of 13 0.5 mg/mL MTT was added, and the cells were incubated at 37 C for 4 h. The absorbance was measured at 570 nm. The data are expressed as the percentage of viable cells compared to the 1% DMSO-treated control [2]. 2.8. Treatment of HaCaT Cells with M. furfur in the Presence or Absence of Citral HaCaT cells were infected with M. furfur at a ratio of 1:20 or 1:30 (HaCaT cells:yeasts), and treated with or without citral for 24 and 48 h. After treatment, cells were washed with PBS and stained with May–Grunwald and Giemsa stain, then examined under a light microscope. The percentage invasiveness was determined by counting the HaCaT cells that the M. furfur yeast had penetrated. The infected cells were treated with or without DMEM containing 0.25 g/mL KTZ for 4 h at 37 C. After this period, cells were scraped and diluted in PBS and plated on mLNA plates. The plates were then incubated for 72 h at 30 C, and colonies were counted (M1 cfu/mL). Yeast cells treated with KTZ were also counted (M0 cfu/mL). The adhesion percentage was then calculated as % adhesion = (M0 M1/M0) *100 [29]. 2.9. ELISA Analysis For measuring the secreted IL-6, IL-10, TNF-, TGF- , and TSLP, cell culture super- natants were collected and tested using the human cytokine ELISA kit (Invitrogen, CA, USA; TSLP from eBioscience) according to the manufacturer ’s instruction. 2.10. RNA Extraction and RT-PCR Analysis Total RNAs were isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and RNeasy Mini Kit (Qiagen, Germantown, MD, USA) from the HaCaT cells treated and not treated with M. furfur, then cDNA was synthesized with the random hexamer primers using the RevertAid H Minus First Stand cDNA Synthesis Kit (Thermo Scientiﬁc, Madison, WI, USA). Real-time RT-PCR (ABI Step One real-time PCR system, Foster City, CA, USA) was used to analyze the mRNA expression of the target genes with the Maxima SYBR Green qPCR master mix kit (Fermentas, Waltham, MA, USA) according to the manufacturer ’s protocol. Table 1 summarizes the primer sets information and reaction conditions. Table 1. Primer sets for real-time RT-PCR. Gene Sense and Anti-Sense Sequence Size (bp) 0 0 5 -TGTCTTGTGACCGCAATGGT-3 TLR2 101 0 0 5 -TGTTGGACAGGTCAAGGCTTT-3 0 0 5 -TAGCAATCGGCCACATTGCC-3 TSLP 145 0 0 5 -CTGAGTTTCCGAATAGCCTG-3 0 0 5 -ATCAGCCATGAGGGTCTTGT-3 HBD-2 172 0 0 5 -GAGACCACAGGTGCCAATTT-3 0 0 5 -TGAACGGGAAGCTCACTGG-3 GAPDH 307 5 -TCCACCACCCTGTTGCTGTA-3 TLR2, Toll-like receptor 2 [2]; TSLP, thymic stromal lymphopoietin [30]; HBD-2, human beta-defensin 2 [31]; GAPDH, glyceraldehyde-3-phosphate dehydrogenase [32]. 2.11. Statistical Analysis Each experiment was performed at least in triplicate. Results were analyzed for statistical signiﬁcance by unpaired Student’s t-test using SigmaPlot v14. A p-value < 0.05 was considered statistically signiﬁcant. 3. Results 3.1. Antifungal Activity of Citral against M. furfur The MIC and MFC of citral against M. furfur were analyzed in triplicate using the microdilution method to determine the fungicidal activities of the citral. KTZ was used as a positive control. The MIC and MFC of citral were 200 g/mL and 300 g/mL, and Processes 2022, 10, x FOR PEER REVIEW 5 of 14 Processes 2022, 10, x FOR PEER REVIEW 5 of 14 Each experiment was performed at least in triplicate. Results were analyzed for sta- Each experiment was performed at least in triplicate. Results were analyzed for sta- tistical significance by unpaired Student’s t-test using SigmaPlot v14. A p-value < 0.05 was tistical significance by unpaired Student’s t-test using SigmaPlot v14. A p-value < 0.05 was considered statistically significant. considered statistically significant. 3. Results 3. Results 3.1. Antifungal Activity of Citral against M. furfur 3.1. Antifungal Activity of Citral against M. furfur The MIC and MFC of citral against M. furfur were analyzed in triplicate using the The MIC and MFC of citral against M. furfur were analyzed in triplicate using the microdilution method to determine the fungicidal activities of the citral. KTZ was used as Processes 2022, 10, 802 5 of 13 microdilution method to determine the fungicidal activities of the citral. KTZ was used as a positive control. The MIC and MFC of citral were 200 μg/mL and 300 μg/mL, and KTZ a positive control. The MIC and MFC of citral were 200 μg/mL and 300 μg/mL, and KTZ were 0.13 μg/mL and 0.25 μg/mL, respectively (Table 2). The MIC and MFC of citral were 0.13 μg/mL and 0.25 μg/mL, respectively (Table 2). The MIC and MFC of citral against M. furfur were higher than those of known antifungal agents such as KTZ. The KTZ were 0.13 g/mL and 0.25 g/mL, respectively (Table 2). The MIC and MFC of citral against M. furfur were higher than those of known antifungal agents such as KTZ. The time–killing curves showed that the fungicidal activity of citral against M. furfur de- against M. furfur were higher than those of known antifungal agents such as KTZ. The time–killing curves showed that the fungicidal activity of citral against M. furfur de- pended on its concentration (Figure 1). Citrate exhibited rapid killing during the first 2–4 time–killing curves showed that the fungicidal activity of citral against M. furfur depended pended on its concentration (Figure 1). Citrate exhibited rapid killing during the first 2–4 h of the assay above MFC. Citral treatment also significantly increased the proportions of on its concentration (Figure 1). Citrate exhibited rapid killing during the ﬁrst 2–4 h of the h of the assay above MFC. Citral treatment also significantly increased the proportions of yeast cells to mycelial forms, by approximately 2.6-fold (Figure 2). assay above MFC. Citral treatment also signiﬁcantly increased the proportions of yeast cells yeast cells to mycelial forms, by approximately 2.6-fold (Figure 2). to mycelial forms, by approximately 2.6-fold (Figure 2). Table 2. The antifungal activity of citral and ketoconazole (KTZ) against M. furfur. Table 2. The antifungal activity of citral and ketoconazole (KTZ) against M. furfur. Table 2. The antifungal activity of citral and ketoconazole (KTZ) against M. furfur. MIC (μg/mL) MFC (μg/mL) MIC (μg/mL) MFC (μg/mL) MIC (g/mL) MFC (g/mL) Citral 200 ± 15 300 ± 25 Citral 200 ± 15 300 ± 25 Citral 200 15 300 25 Ket Ketoconazole oconazole 0.1 0.13 3 ± 0.05 0.05 0.20.25 5 ± 0.09 0.09 Ketoconazole 0.13 ± 0.05 0.25 ± 0.09 All results are the means of three determinations. All results are the means of three determinations. All results are the means of three determinations. Figure 1. Time–killing curves of the citral against Malassezia furfur. M. furfur was treated with dif- Figure 1. Time–killing curves of the citral against Malassezia furfur. M. furfur was treated with Figure 1. Time–killing curves of the citral against Malassezia furfur. M. furfur was treated with dif- ferent concentrations of citral in the mDixon medium. After 1, 2, 3, and 4 h, the yeasts were spread different concentrations of citral in the mDixon medium. After 1, 2, 3, and 4 h, the yeasts were spread ferent concentrations of citral in the mDixon medium. After 1, 2, 3, and 4 h, the yeasts were spread on the mDixon plates and incubated for 72 h at 30 °C. The colony-forming units were counted and on the mDixon plates and incubated for 72 h at 30 C. The colony-forming units were counted and on the mDixon plates and incubated for 72 h at 30 °C. The colony-forming units were counted and compared to control plates. compared to control plates. compared to control plates. Figure 2. Citral increased the proportions of yeast cells to mycelial forms. M. furfur was treated with Figure 2. Citral increased the proportions of yeast cells to mycelial forms. M. furfur was treated with Figure 2. Citral increased the proportions of yeast cells to mycelial forms. M. furfur was treated with different concentrations of citral for 48 h at 30 °C. Yeast cells were stained with the LPCB method different concentrations of citral for 48 h at 30 °C. Yeast cells were stained with the LPCB method different concentrations of citral for 48 h at 30 C. Yeast cells were stained with the LPCB method and and examined at 1000× magnification using a light microscope. and examined at 1000× magnification using a light microscope. examined at 1000 magniﬁcation using a light microscope. 3.2. Citral Induced Apoptosis in M. furfur 3.2.1. Phosphatidylserine Externalization In combination with membrane-impermeable dye, PI, Annexin V-FITC stain, which 2+ binds to phosphatidylserine with high afﬁnity in the presence of Ca , was used to deter- mine the citral-induced apoptosis in M. furfur. As shown in Figure 3, citral-treated M. furfur cells showed similar phosphatidylserine externalizations to H O -treated M. furfur cells in 2 2 which H O was an inducer of apoptosis in the yeast cells. 2 2 Processes 2022, 10, x FOR PEER REVIEW 6 of 14 3.2. Citral Induced Apoptosis in M. furfur 3.2.1. Phosphatidylserine Externalization In combination with membrane-impermeable dye, PI, Annexin V-FITC stain, which 2+ binds to phosphatidylserine with high affinity in the presence of Ca , was used to deter- mine the citral-induced apoptosis in M. furfur. As shown in Figure 3, citral-treated M. fur- Processes 2022, 10, 802 6 of 13 fur cells showed similar phosphatidylserine externalizations to H2O2-treated M. furfur cells in which H2O2 was an inducer of apoptosis in the yeast cells. Figure 3. Effect of citral on externalization of phosphatidylserine at the cytoplasmic membrane. Figure 3. Effect of citral on externalization of phosphatidylserine at the cytoplasmic membrane. Phosphatidylserine externalizations were determined by Annexin V-FITC and PI staining in citral- Phosphatidylserine externalizations were determined by Annexin V-FITC and PI staining in citral-or or H2O2-treated M. furfur cells using a flow cytometer. (A) Control, (B) 10 mM H2O2 as positive con- H O -treated M. furfur cells using a ﬂow cytometer. (A) Control, (B) 10 mM H O as positive control, 2 2 2 2 trol, (C) 300 μg/mL citral, and (D) 600 μg/mL citral. (C) 300 g/mL citral, and (D) 600 g/mL citral. 3.2.2. DNA Fragmentation 3.2.2. DNA Fragmentation DNA fragmentation is one of the late apoptotic phenotypes. To investigate whether DNA fragmentation is one of the late apoptotic phenotypes. To investigate whether citral induces the late stage of apoptosis in M. furfur, we evaluated DNA fragmentation citral induces the late stage of apoptosis in M. furfur, we evaluated DNA fragmentation using TUNEL assay and flow cytometry. TUNEL assay is a standard method for detecting using TUNEL assay and ﬂow cytometry. TUNEL assay is a standard method for detecting apoptotic DNA cleavage in individual nuclei by labeling the fluorescent dUTP at the 3′- apoptotic DNA cleavage in individual nuclei by labeling the ﬂuorescent dUTP at the 3 -OH Processes 2022, 10, x FOR PEER REVIEW 7 of 14 OH ends of DNA. We found that M. furfur cells exposed to citral and H2O2 showed in- ends of DNA. We found that M. furfur cells exposed to citral and H O showed increased 2 2 creased fluorescence intensity compared to untreated cells (Figure 4). ﬂuorescence intensity compared to untreated cells (Figure 4). Figure 4. Effect of citral-induced DNA fragmentation in M. furfur cells. Cells were treated with citral Figure 4. Effect of citral-induced DNA fragmentation in M. furfur cells. Cells were treated with or H2O2 for (A) 3 h and (B) 24 h. Cells were stained with TUNEL staining and analyzed using flow citral or H O for (A) 3 h and (B) 24 h. Cells were stained with TUNEL staining and analyzed using 2 2 cytometry. ﬂow cytometry. 3.2.3. Metacaspase Activation The activation of metacaspase plays a vital role in apoptosis. Citral-treated M. furfur cells were incubated with FITC-labeled VAD-FMK to monitor the metacaspase activation. The FITC-labeled caspase inhibitor VAD-FMK, a cell-permeable fluorescent maker, binds specifically to the active center of metazoan caspases in apoptotic cell. As shown in Figure 5, cells treated with citral showed increased fluorescence intensity, consistent with the positive control cells treated with H2O2. These results suggest that citral induces the bio- chemical feature of apoptosis in M. furfur cells, including membrane depolarization, DNA fragmentation, and metacaspase activation. Figure 5. Effect of citral on the activity of metacaspase in M. furfur cells. Cells were stained with FITC-VAD-FMK, and analyzed using a flow cytometer. (A) 10 mM H2O2, (B) 300 μg/mL citral, and (C) 600 μg/mL citral. Processes 2022, 10, x FOR PEER REVIEW 7 of 14 Figure 4. Effect of citral-induced DNA fragmentation in M. furfur cells. Cells were treated with citral Processes 2022, 10, 802 7 of 13 or H2O2 for (A) 3 h and (B) 24 h. Cells were stained with TUNEL staining and analyzed using flow cytometry. 3.2.3. Metacaspase Activation 3.2.3. Metacaspase Activation The activation of metacaspase plays a vital role in apoptosis. Citral-treated M. furfur The activation of metacaspase plays a vital role in apoptosis. Citral-treated M. furfur cells were incubated with FITC-labeled VAD-FMK to monitor the metacaspase activation. cells were incubated with FITC-labeled VAD-FMK to monitor the metacaspase activation. The FITC-labeled caspase inhibitor VAD-FMK, a cell-permeable fluorescent maker, binds The FITC-labeled caspase inhibitor VAD-FMK, a cell-permeable ﬂuorescent maker, binds specifically to the active center of metazoan caspases in apoptotic cell. As shown in Figure speciﬁcally to the active center of metazoan caspases in apoptotic cell. As shown in 5, cells treated with citral showed increased fluorescence intensity, consistent with the Figure 5, cells treated with citral showed increased ﬂuorescence intensity, consistent with positive control cells treated with H2O2. These results suggest that citral induces the bio- the positive control cells treated with H O . These results suggest that citral induces the 2 2 chemica biochemical l feature o featur f apopt e of apoptosis osis in M. furfu in M. r cel furfur ls, incl cells, udiincl ng membran uding membr e depol ane ardepolari ization, DNA zation, fra DNA gment fragmentation, ation, and meand tacasp metacaspase ase activatiactivation. on. Processes 2022, 10, x FOR PEER REVIEW 8 of 14 Figure 5. Effect of citral on the activity of metacaspase in M. furfur cells. Cells were stained with Figure 5. Effect of citral on the activity of metacaspase in M. furfur cells. Cells were stained with FITC-VAD-FMK, and analyzed using a ﬂow cytometer. (A) 10 mM H O (B) 300 g/mL citral, and 2 2, FITC-VAD-FMK, and analyzed using a flow cytometer. (A) 10 mM H2O2, (B) 300 μg/mL citral, and (C) 600 g/mL citral. (C) 600 μg/mL citral. 3.3. Citral Can Decrease the Adhesiveness and Invasiveness of M. furfur to Human 3.3. Citral Can Decrease the Adhesiveness and Invasiveness of M. furfur to Human Keratinocytes Keratinocytes at Sub-MIC at Sub-MIC 3.3.1. The Cytotoxic Effect of Citral on HaCaT Cells 3.3.1. The Cytotoxic Effect of Citral on HaCaT Cells For determine the cytotoxic effects in HaCaT cells, cell viabilities were measured us- For determine the cytotoxic effects in HaCaT cells, cell viabilities were measured ing an MTT assay. After treatment with different concentrations of citral, the viability per- using an MTT assay. After treatment with different concentrations of citral, the viability centages of HaCaT cells were >98% (Figure 6). These results demonstrate that citral has no percentages of HaCaT cells were >98% (Figure 6). These results demonstrate that citral has cyt no o cytotoxic toxic effe ef ctfect in H in aHaCaT CaT cellcells s at tat he ant the antifungal ifungal conc concentrations. entrations. Figure 6. Cell viability of HaCaT cells to citral. HaCaT cells were treated with different concentrations Figure 6. Cell viability of HaCaT cells to citral. HaCaT cells were treated with different concentra- of citral for (A) 48 h and (B) 72 h. An MTT assay was used to determine the cell viabilities. tions of citral for (A) 48 h and (B) 72 h. An MTT assay was used to determine the cell viabilities. 3.3.2. The Inhibitory Effect of Citral on M. furfur Adhesion to HaCaT Cells 3.3.2. The Inhibitory Effect of Citral on M. furfur Adhesion to HaCaT Cells For determining whether citral could interfere with M. furfur invasion, the HaCaT For determining whether citral could interfere with M. furfur invasion, the HaCaT cells were treated with different concentrations of citral and infected with M. furfur (1:20 or cells were treated with different concentrations of citral and infected with M. furfur (1:20 1:30, cells:yeasts) for 24 h. We can found that at low concentrations (25, 50 g/mL), citral or 1:30, cells:yeasts) for 24 h. We can found that at low concentrations (25, 50 μg/mL), citral can inhibit the adhesion of M. furfur to HaCaT cells and the inhibition was more apparent can inhibit the adhesion of M. furfur to HaCaT cells and the inhibition was more apparent at higher concentrations (100, 200 g/mL) compared with the control (Figure 7). at higher concentrations (100, 200 μg/mL) compared with the control (Figure 7). Figure 7. The effect of citral against M. furfur adherence on HaCaT cells. HaCaT cells were cultured with citral and M. furfur at a ratio of 1:20 or 1:30 (HaCaT cells:yeasts). After 24 h, the infected cells were treated with or without DMEM containing 0.25 μg/mL ktz for 4 h. The adhesion percentage was then calculated as % adhesion = (M0 − M1/M0) *·100. (M1,CFU of treated with various citral concentrations; M0, CFU of treated with ktz). Adherence differences were determined using the unpaired Student’s t-test (* p < 0.05). Processes 2022, 10, x FOR PEER REVIEW 8 of 14 3.3. Citral Can Decrease the Adhesiveness and Invasiveness of M. furfur to Human Keratinocytes at Sub-MIC 3.3.1. The Cytotoxic Effect of Citral on HaCaT Cells For determine the cytotoxic effects in HaCaT cells, cell viabilities were measured us- ing an MTT assay. After treatment with different concentrations of citral, the viability per- centages of HaCaT cells were >98% (Figure 6). These results demonstrate that citral has no cytotoxic effect in HaCaT cells at the antifungal concentrations. Figure 6. Cell viability of HaCaT cells to citral. HaCaT cells were treated with different concentra- tions of citral for (A) 48 h and (B) 72 h. An MTT assay was used to determine the cell viabilities. 3.3.2. The Inhibitory Effect of Citral on M. furfur Adhesion to HaCaT Cells For determining whether citral could interfere with M. furfur invasion, the HaCaT cells were treated with different concentrations of citral and infected with M. furfur (1:20 or 1:30, cells:yeasts) for 24 h. We can found that at low concentrations (25, 50 μg/mL), citral Processes 2022, 10, 802 8 of 13 can inhibit the adhesion of M. furfur to HaCaT cells and the inhibition was more apparent at higher concentrations (100, 200 μg/mL) compared with the control (Figure 7). Figure 7. The effect of citral against M. furfur adherence on HaCaT cells. HaCaT cells were cultured Figure 7. The effect of citral against M. furfur adherence on HaCaT cells. HaCaT cells were cultured with citral and M. furfur at a ratio of 1:20 or 1:30 (HaCaT cells:yeasts). After 24 h, the infected cells with citral and M. furfur at a ratio of 1:20 or 1:30 (HaCaT cells:yeasts). After 24 h, the infected cells were treated with or without DMEM containing 0.25 μg/mL ktz for 4 h. The adhesion percentage were treated with or without DMEM containing 0.25 g/mL ktz for 4 h. The adhesion percentage was then calculated as % adhesion = (M0 − M1/M0) *·100. (M1,CFU of treated with various citral Processes 2022, 10, x FOR PEER REVIEW was then calculated as % adhesion = (M0 M1/M0) *100. (M1,CFU of treated with various 9 of citral 14 concentrations; M0, CFU of treated with ktz). Adherence differences were determined using the concentrations; M0, CFU of treated with ktz). Adherence differences were determined using the unpaired Student’s t-test (* p < 0.05). unpaired Student’s t-test (* p < 0.05). 3.3.3. The Inhibitory Effect of Citral on M. furfur Invasiveness into HaCaT Cells 3.3.3. The Inhibitory Effect of Citral on M. furfur Invasiveness into HaCaT Cells The invasion of M. furfur to HaCaT cells was monitored using May−Grunwald and The invasion of M. furfur to HaCaT cells was monitored using MayGrunwald and Giemsa staining. Some HaCaT cells showed yeast engulfment in the negative-control Giemsa staining. Some HaCaT cells showed yeast engulfment in the negative-control group group and low-concentration citral-treatment groups (Figure 8A). Cell invasiveness of and low-concentration citral-treatment groups (Figure 8A). Cell invasiveness of 15% and 15% and 30% were determined at HaCaT cells to yeasts ratios of 1:20 and 1:30 (Figure 8B). 30% were determined at HaCaT cells to yeasts ratios of 1:20 and 1:30 (Figure 8B). Like the Like the adhesion, citral reduced the invasion of M. furfur to HaCaT cells (Figure 8C). adhesion, citral reduced the invasion of M. furfur to HaCaT cells (Figure 8C). Figure 8. The effect of citral against M. furfur invasion on HaCaT cells. HaCaT cells treated with Figure 8. The effect of citral against M. furfur invasion on HaCaT cells. HaCaT cells treated with citral and M. furfur at a ratio of 1:20 and 1:30 (HaCaT cells:yeasts) for 24 and 48 h. (A) HaCaT cells citral and M. furfur at a ratio of 1:20 and 1:30 (HaCaT cells:yeasts) for 24 and 48 h. (A) HaCaT cells were stained with the May–Grunwald and Giemsa method after incubation with yeast. Some cells were stained with the May–Grunwald and Giemsa method after incubation with yeast. Some cells show yeast engulfment. (B, C) Yeasts were taken up into HaCaT cells after being treated different show yeast engulfment. (B,C) Yeasts were taken up into HaCaT cells after being treated different concentrations of citral for 24 and 48 h. The percentage of invasiveness was determined by counting concentrations of citral for 24 and 48 h. The percentage of invasiveness was determined by counting the HaCaT cells that the M. furfur yeast had penetrated. The negative control group (cells:yeasts the HaCaT cells that the M. furfur yeast had penetrated. The negative control group (cells:yeasts ratio ratio of 1:30) was set as 100%. Differences in adherence were determined using the unpaired Stu- of 1:30) was set as 100%. Differences in adherence were determined using the unpaired Student’s dent’s t-test (* p < 0.05). t-test (* p < 0.05). 3.4. Citral Can Modulate the Immune Response of HUMAN keratinocytes and Interfere with M. furfur Infection 3.4.1. TLR2, HBD-2, and TSLP Gene Expression We next examined the effect of citral on the mRNA expression of TLR2, HBD-2, and TSLP by real-time RT-PCR in the M. furfur-infected HaCaT cells. HaCaT cells were treated with M. furfur (cells:yeasts of 1:30) at different citral sub-MICs. The TLR2 mRNA expres- sion was 30-fold higher in M. furfur-treated HaCaT cells at 8 h treatment. In contrast, when cells were cotreated with M. furfur and citral, the increased TLR2 mRNA expression was significantly downregulated by citral (Figure 9A), and the TLR2 mRNA expression down- regulations were also found after 24 h treatment (Figure 9B). Moreover, both citral and M. furfur induced HBD-2 production, with the strongest effect occurring in M. furfur-infected cells after 24 h treatment (Figure 10). Finally, we tested the effect of citral on TLSP mRNA expression. The TSLP mRNA expression increased in the M. furfur-infected cells, and citral had a more apparent effect on TSLP transcript after 24 h treatment (Figure 11). These re- sults demonstrate that citral efficiently inhibits the M. furfur-induced TLR2 mRNA expres- sion and enhances HBD-2 and TSLP mRNA expression in HaCaT cells. Processes 2022, 10, 802 9 of 13 3.4. Citral Can Modulate the Immune Response of HUMAN keratinocytes and Interfere with M. furfur Infection 3.4.1. TLR2, HBD-2, and TSLP Gene Expression We next examined the effect of citral on the mRNA expression of TLR2, HBD-2, and TSLP by real-time RT-PCR in the M. furfur-infected HaCaT cells. HaCaT cells were treated with M. furfur (cells:yeasts of 1:30) at different citral sub-MICs. The TLR2 mRNA expression was 30-fold higher in M. furfur-treated HaCaT cells at 8 h treatment. In contrast, when cells were cotreated with M. furfur and citral, the increased TLR2 mRNA expression was signiﬁcantly downregulated by citral (Figure 9A), and the TLR2 mRNA expression downregulations were also found after 24 h treatment (Figure 9B). Moreover, both citral and M. furfur induced HBD-2 production, with the strongest effect occurring in M. furfur- infected cells after 24 h treatment (Figure 10). Finally, we tested the effect of citral on TLSP mRNA expression. The TSLP mRNA expression increased in the M. furfur-infected cells, and citral had a more apparent effect on TSLP transcript after 24 h treatment (Figure 11). Processes 2022, 10, x FOR PEER REVIEW 10 of 14 These results demonstrate that citral efﬁciently inhibits the M. furfur-induced TLR2 mRNA Processes 2022, 10, x FOR PEER REVIEW 10 of 14 expression and enhances HBD-2 and TSLP mRNA expression in HaCaT cells. Figure 9. The gene expression of TLR2 in citral against M. furfur-infected HaCaT cells. HaCaT cells Figure 9. The gene expression of TLR2 in citral against M. furfur-infected HaCaT cells. HaCaT cells Figure 9. The gene expression of TLR2 in citral against M. furfur-infected HaCaT cells. HaCaT cells were cotreated with citral and M. furfur at a cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. Cells were cotreated with citral and M. furfur at a cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. Cells were cotreated with citral and M. furfur at a cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. Cells were scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine the TLR2 were scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine the TLR2 were scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine the TLR2 mRNA expression. All values are expressed as mean ± SD. Differences in the expression of TLR2 mRNA expression. All values are expressed as mean SD. Differences in the expression of TLR2 mRNA expression. All values are expressed as mean ± SD. Differences in the expression of TLR2 were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with non-treat- were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with non-treatment were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with non-treat- ment control, # means comparison with M. furfur-infected cells.). ment control, # means comparison with M. furfur-infected cells.). control, # means comparison with M. furfur-infected cells.). Figure 10. The gene expression of HBD-2 in citral against M. furfur-infected HaCaT cells. HaCaT Figure 10. The gene expression of HBD-2 in citral against M. furfur-infected HaCaT cells. HaCaT Figure 10. The gene expression of HBD-2 in citral against M. furfur-infected HaCaT cells. HaCaT cells were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. cells were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. cells were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. Cells were scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine the Cells were scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine the HBD-2 mRNA Cells were scrap-harvested expression. Aland l values quantitatively are expressed as mean ± SD analyzed using real-time . Differences in the RT-PCR to expression determine of the HBD-2 mRNA expression. All values are expressed as mean ± SD. Differences in the expression of HBD-2 were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with non- HBD-2 mRNA expression. All values are expressed as mean SD. Differences in the expression HBD-2 were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with non- treatment control, # means comparison with M. furfur-infected cells.). of HBD-2 were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with treatment control, # means comparison with M. furfur-infected cells.). non-treatment control, # means comparison with M. furfur-infected cells.). Figure 11. The gene expression of TSLP in citral against M. furfur-infected HaCaT cells. HaCaT cells Figure 11. The gene expression of TSLP in citral against M. furfur-infected HaCaT cells. HaCaT cells were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for 24 h. Cells were scrap- were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for 24 h. Cells were scrap- harvested and quantitatively analyzed using real-time RT-PCR to determine TSLP mRNA expres- harvested and quantitatively analyzed using real-time RT-PCR to determine TSLP mRNA expres- sion. All values are expressed as mean ± SD. Differences in TSLP expression were determined using sion. All values are expressed as mean ± SD. Differences in TSLP expression were determined using Processes 2022, 10, x FOR PEER REVIEW 10 of 14 Figure 9. The gene expression of TLR2 in citral against M. furfur-infected HaCaT cells. HaCaT cells were cotreated with citral and M. furfur at a cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. Cells were scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine the TLR2 mRNA expression. All values are expressed as mean ± SD. Differences in the expression of TLR2 were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with non-treat- ment control, # means comparison with M. furfur-infected cells.). Figure 10. The gene expression of HBD-2 in citral against M. furfur-infected HaCaT cells. HaCaT cells were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. Cells were scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine the HBD-2 mRNA expression. All values are expressed as mean ± SD. Differences in the expression of Processes 2022, 10, 802 10 of 13 HBD-2 were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with non- treatment control, # means comparison with M. furfur-infected cells.). Processes 2022, 10, x FOR PEER REVIEW 11 of 14 Figure 11. The gene expression of TSLP in citral against M. furfur-infected HaCaT cells. HaCaT cells Figure 11. The gene expression of TSLP in citral against M. furfur-infected HaCaT cells. HaCaT were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for 24 h. Cells were scrap- cells were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for 24 h. Cells were harvested and quantitatively analyzed using real-time RT-PCR to determine TSLP mRNA expres- scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine TSLP mRNA unpaired Student’s t-test (* p < 0.05). (* means comparison with non-treatment control, # means sion. All values are expressed as mean ± SD. Differences in TSLP expression were determined using expression. All values are expressed as mean SD. Differences in TSLP expression were determined comparison with M. furfur-infected cells.). using unpaired Student’s t-test (* p < 0.05). (* means comparison with non-treatment control, # means comparison with M. furfur-infected cells.). 3.4.2. Cytokine Expression Assay 3.4.2. Cytokine Expression Assay To determine how citral interferes with M. furfur, we examined the expressions of proinflammatory cytokines IL-6 and TNF-α, and anti-inflammatory cytokines IL-10 and To determine how citral interferes with M. furfur, we examined the expressions of TGF-β using ELISA. IL-6 levels increased in M. furfur-infected HaCaT cells after 24 h of proinﬂammatory cytokines IL-6 and TNF-, and anti-inﬂammatory cytokines IL-10 and trea TGF- tment. In contra using ELISA. st, IL-6 a signi levels fica incr nt decrea eased s in e in M. IL-6 furfur level w -infected as observed when t HaCaT cells afterhe cells 24 h of were trea treatment. ted In wi contrast, th citral ( aFsigniﬁcant igure 12). The resu decreaselts cle in IL-6 arly demonstr level was observed ated that c when itral the inhibits cells the proi were treated nflamwith mtory cytokine citral (FigurIL-6 expre e 12). The ssion results ind clearly uced by demonstrated M. furfur in that HaC citral aT cinhibits ells. How- the ever, expre proinﬂammtory ssion d cytokine ifference IL-6 s in TNF expression -α, IL-1 induced 0, and T by GF- M.β were n furfur inoHaCaT t detectable cells. (dat However a not , shown). expression differences in TNF-, IL-10, and TGF- were not detectable (data not shown). Figure 12. IL-6 expression in citral against M. furfur-infected HaCaT cells. The secretion of IL-6 in- Figure 12. IL-6 expression in citral against M. furfur-infected HaCaT cells. The secretion of IL-6 duced by the M. furfur-infected HaCaT cells was measured using ELISA. Cell supernatants were induced by the M. furfur-infected HaCaT cells was measured using ELISA. Cell supernatants were collected at 24 h after infection with M. furfur in the presence or absence of citral. The negative con- collected at 24 h after infection with M. furfur in the presence or absence of citral. The negative trol was the non-treated cells. The results are means of triplicate experiments. All values are ex- control was the non-treated cells. The results are means of triplicate experiments. All values are pressed as mean ± SD. Differences in IL-6 expression were determined using unpaired Student’s t- expressed as mean SD. Differences in IL-6 expression were determined using unpaired Student’s test (* p < 0.05). (* means comparison with non-treatment control, # means comparison with M. fur- t-test (* p < 0.05). (* means comparison with non-treatment control, # means comparison with M. fur-infected cells.). furfur-infected cells.). 4. Discussion 4. Discussion Malassezia spp. yeasts are dimorphic fungi and normal fungal skin flora in humans Malassezia spp. yeasts are dimorphic fungi and normal fungal skin ﬂora in humans and other warm-blooded animals. They can cause skin-related diseases through different and other warm-blooded animals. They can cause skin-related diseases through different mechanisms. Killing bacteria or bacterial inhibiting was the core concept of antibiotic de- velopment and an essential milestone in medicinal chemistry. Destroying critical compo- nents during bacterial growth is an efficient antibacterial strategy. Although clinical stud- ies have confirmed the azoles such as ketoconazole (KTZ) effectively treat Malassezia-re- lated skin diseases, the long-term safety of antifungal drugs remains unknown [7]. Tox- icity, low efficacy, and drug resistance of the antifungal drugs have limited their clinical usage. Besides, they are costly when an extended treatment time is necessary and many patients leave the therapy before being cured. One way to prevent antibiotic resistance of pathogenic species is to find and use new compounds [3,21]. Both mycelial and yeast forms have been found in several diseases believed to be caused by M. furfur. Our study showed that citral has lower antifungal activity below sub- MIC but can block the yeast to mycelia transformation of M. furfur, although the mecha- nism and effect remained unclear. In the early phases of apoptosis, phosphatidylserine is translocated from the inner leaflet of the plasma membrane bilayers to the outer leaflet. Phosphatidylserine exposure generally precedes DNA fragmentation and nuclear condensation. A putative caspase (metacaspase) has been shown to be involved in apoptosis. Our results suggested that Processes 2022, 10, 802 11 of 13 mechanisms. Killing bacteria or bacterial inhibiting was the core concept of antibiotic devel- opment and an essential milestone in medicinal chemistry. Destroying critical components during bacterial growth is an efﬁcient antibacterial strategy. Although clinical studies have conﬁrmed the azoles such as ketoconazole (KTZ) effectively treat Malassezia-related skin diseases, the long-term safety of antifungal drugs remains unknown [7]. Toxicity, low efﬁ- cacy, and drug resistance of the antifungal drugs have limited their clinical usage. Besides, they are costly when an extended treatment time is necessary and many patients leave the therapy before being cured. One way to prevent antibiotic resistance of pathogenic species is to ﬁnd and use new compounds [3,21]. Both mycelial and yeast forms have been found in several diseases believed to be caused by M. furfur. Our study showed that citral has lower antifungal activity below sub-MIC but can block the yeast to mycelia transformation of M. furfur, although the mechanism and effect remained unclear. In the early phases of apoptosis, phosphatidylserine is translocated from the inner leaﬂet of the plasma membrane bilayers to the outer leaﬂet. Phosphatidylserine exposure generally precedes DNA fragmentation and nuclear condensation. A putative caspase (metacaspase) has been shown to be involved in apoptosis. Our results suggested that citral at lower concentrations induces apoptosis in M. furfur yeast cells and necrosis at higher concentrations. The yeast apoptosis process has been found in common clinical yeast such as C. albican, but remains unclear in M. furfur [7,32–34]. In the current experiment, we chose the HaCaT cell line to examine the effect of citral on M. furfur-infected cells. At ﬁrst, M. furfur cultured in the mDixon medium barely infected the HaCaT cells. Malassezia spp. has cell walls with a very thick multi-layered structure, and the lipid compositions alter when cultivated in the different medium. The lipid layer of Malassezia spp. plays an essential role in modulating proinﬂammatory cytokine production by keratinocytes [35]. Therefore, we changed the mDixon medium to mLNA medium with higher lipid content to cultivate M. furfur for cell infection experiments and found that the infection rate of mLNA-cultured M. furfur in HaCaT cells increased signiﬁcantly compared to that of mDixon-cultured M. furfur. Toll-like receptors (TLRs) are crucial players in the innate immune responses to micro- bial invaders, TLRs and -defensins are crucial elements in the innate immune response against bacteria, fungi, and viruses affecting the human skin and TLR2 is one of the main receptors on the cell surface that recognizes fungus [2]. Our study demonstrated that citral downregulates TLR2 gene expression in M. furfur-infected keratinocytes. In addition, citral also increased the expression of HBD-2 antimicrobial peptides in HaCaT cells. The antimicrobial peptides directly bind to the cell membranes of microorganisms and alter their permeability resulting in a bacteriostatic effect. Citral may stimulate HaCaT cells to secrete antimicrobial peptides, which inhibit M. furfur growth. The binding of HBD-2 may also prevent the binding of M. furfur to cells due to steric structural barriers. In addition to recognizing foreign pathogens, TLR2 also initiates host innate immunity and affects subsequent adaptive immune responses. In recent studies, Malassezia stimulates human keratinocytes to produce cytokines and inﬂammatory molecules [20,36]. We found that citral inhibits IL-6 production in M. furfur-infected HaCaT cells. A previous study has shown that citral has an anti-inﬂammatory ability on lipopolysaccharide (LPS)-induced RAW 264.7 mouse macrophages and can inhibit the inﬂammatory cytokines molecules such as IL-6 and IL-10 [37]. Because the TLR2 precedes IL-6 and IL-8 pathways, it is reasonable to speculate that citral inhibits the TLR2 expression in HaCaT cells, resulting in a decrease in IL-6 secretion. Further research is required. 5. Conclusions In conclusion, citral has antifungal activity at high concentrations and can block morphogenesis of M. furfur at low concentrations. Citral decreases the infection of M. furfur through modulation of the keratinocyte immune response. Citral modulates the M. furfur infection in HaCaT cells by inducing the secretion of HBD-2 antimicrobial peptides. In Processes 2022, 10, 802 12 of 13 addition, citral inhibits TLR2 expression and cell recognition of M. furfur. Citral also affects the expression of M. furfur surface adhesion factor, reducing the ability of M. furfur to adhere and aggregate. Our ﬁndings suggest that citral is a potential drug component to be formulated in therapeutics for M. furfur-associated human skin diseases. Author Contributions: W.-L.L. and M.-H.L. conceived and planned the experiments. Y.-T.L. and Y.-S.L. carried out the experiments. W.-L.L. and M.-H.L. took the lead in writing the manuscript. All authors provided critical feedback and helped shape the research, analysis, and manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by grants from the Chung Shan Medical University Founda- tion, Taiwan (No. CSMU/PU-103-2). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors would like to thank El-Wui Lo for improving the use of English in the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Theelen, B.; Cafarchia, C.; Gaitanis, G.; Bassukas, I.D.; Boekhout, T.; Dawson, J.T.L. Malassezia ecology, pathophysiology, and treatment. Med. Mycol. 2018, 56, S10–S25. [CrossRef] [PubMed] 2. Ryu, S.; Choi, S.Y.; Acharya, S.; Chun, Y.J.; Gurley, C.; Park, Y.; Armstrong, C.A.; Song, P.I.; Kim, B.J. 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Antifungal activity of coumarin against Candida albicans is related to apoptosis. Front. Cell. Infect. Microbiol. 2019, 8, 445. [CrossRef] 35. Akaza, N.; Akamatsu, H.; Takeoka, S.; Mizutani, H.; Nakata, S.; Matsunaga, K. Increased hydrophobicity in Malassezia species correlates with increased proinﬂammatory cytokine expression in human keratinocytes. Med. Mycol. 2012, 50, 802–810. [CrossRef] [PubMed] 36. Park, H.R.; Oh, J.H.; Lee, Y.J.; Park, S.H.; Lee, Y.W.; Lee, S.; Kang, H.; Kim, J.E. Inﬂammasome-mediated Inﬂammation by Malassezia in human keratinocytes: A comparative analysis with different strains. Mycoses 2021, 64, 292–299. [CrossRef] 37. Bachiega, T.F.; Sforcin, J.M. Lemongrass and citral effect on cytokines production by murine macrophages. J. Ethnopharmacol. 2011, 137, 909–913. 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Abstract

processes Article 1 2 2 1 , 3 , Yi-Tsz Liu , Meng-Hwan Lee , Yin-Shen Lin and Wen-Lin Lai * Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan; 1052039@mail.atri.org.tw Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute, Zhunan Township 35053, Taiwan; mhlee@mail.atri.org.tw (M.-H.L.); chris112783@mail.atri.org.tw (Y.-S.L.) Clinical Laboratory, Chung Shan Medical University Hospital, Taichung 40201, Taiwan * Correspondence: wllai@csmu.edu.tw; Tel.: +886-4-24730022 (ext. 12421) Abstract: The lipophilic yeast Malassezia furfur, is a member of the cutaneous commensal microbiota and is associated with several chronic diseases such as dandruff, pityriasis versicolor, folliculitis, and seborrheic dermatitis, that are often difﬁcult to treat with current therapies. The development of alternatively effective antifungal therapies is therefore of paramount importance. In this study, we investigated the treatment effect of citral on M. furfur. The minimal inhibitory concentration of citral for M. furfur was 200 g/mL, and the minimal fungicidal concentration was 300 g/mL. Citral signiﬁcantly increased the proportion of yeast cells to mycelial forms 2.6-fold. Phosphatidylserine externalization, DNA fragmentation, and metacaspase activation supported a citral-induced apop- tosis in M. furfur. Moreover, citral at sub-minimum inhibitory concentrations reduced the invasion of M. furfur in HaCaT keratinocytes. Finally, we demonstrated that citral inhibited IL-6 and TLR-2 expression and enhanced HBD-2 and TSLP expression in M. furfur-infected HaCaT keratinocytes. These results showed that citral has antifungal activity at high concentrations and can decrease the infection of M. furfur by modulating the keratinocyte immune responses at low concentrations. Our results suggest that citral is a potential candidate for topical therapeutic application for M. furfur-associated human skin diseases. Citation: Liu, Y.-T.; Lee, M.-H.; Lin, Keywords: Malassezia furfur; citral; yeast apoptosis; human keratinocyte; immunomodulation Y.-S.; Lai, W.-L. The Inhibitory Activity of Citral against Malassezia furfur. Processes 2022, 10, 802. https://doi.org/10.3390/pr10050802 1. Introduction Academic Editor: Maurizio Ventre The lipophilic yeast Malassezia furfur is a member of the cutaneous commensal micro- Received: 17 February 2022 biota of human skin, and is found particularly in areas rich in sebaceous gland content. Accepted: 17 April 2022 M. furfur infection can result in several chronic superﬁcial dermatitis such as dandruff, Published: 19 April 2022 pityriasis versicolor, seborrheic dermatitis, folliculitis, and atopic dermatitis. M. furfur is a dimorphic fungus that can alter its morphology from a unicellular yeast form to a Publisher’s Note: MDPI stays neutral mycelial form. M. furfur primarily shows a yeast form in normal conditions and standard with regard to jurisdictional claims in published maps and institutional afﬁl- cultures. However, it transforms into a mycelial form under some stimulations which play iations. a predominant role in the pathogenesis of some diseases, such as pityriasis versicolor [1–5]. Several azoles, such as ketoconazole (KTZ) in solutions, creams, gels, and shampoo forms are used in routine clinical treatment of M. furfur. However, KTZ demonstrates toxicity to mammalian cells and may cause urticarial [6]. M. furfur-related diseases are Copyright: © 2022 by the authors. often refractory to therapy and require extended use of antifungal and anti-inﬂammatory Licensee MDPI, Basel, Switzerland. medications, which may lead to drug resistance [7–9]. Therefore, ﬁnding a safe, effective, This article is an open access article and side-effect-free treatment is required. distributed under the terms and Plants and their derivatives are known sources of a variety of biologically active conditions of the Creative Commons components. They have great potential due to their low cost, low toxicity, and safety. Attribution (CC BY) license (https:// Previous studies have shown that extracts or puriﬁed ingredients from Trigonella foenum- creativecommons.org/licenses/by/ graecum, Asparagus racemosus, Hypericumper foratum, Dittrichia viscosa, and Vitis vinifera had 4.0/). Processes 2022, 10, 802. https://doi.org/10.3390/pr10050802 https://www.mdpi.com/journal/processes Processes 2022, 10, 802 2 of 13 treatment effects on Malassezia spp. infections [10–14]. Cymbopogon citratus, a herb and perennial tropical grass commonly known as lemon grass, is widely used in mid-tropical countries such as Southeast Asia, South America, and Africa as food seasoning and perfume material and as herbal medicines for its analgesic and anti-inﬂammatory properties [15]. Citral (3,7-dimethyl-2,6-octadienal) is the major constituent of Cymbopogon citratus and is classiﬁed as a “generally recognized as safe” (GRAS) substance and is used in food, perfume, and cosmetics and as a pharmaceutical component because of its lemon-like ﬂavor. Citral is a mixture of two isomeric acyclic aldehydes, geranial (trans-citral, citral A) and neral (cis-citral, citral B). Several studies have demonstrated that citral possesses antifungal, antimicrobial, and anti-inﬂammatory activities [16,17]. The antifungal activity exerted by citral has been demonstrated in varied conditions. Recently, it has been shown that citral can destroy the integrity of the cell membrane. Citral could also exert its antifungal effect by inhibiting ergosterol biosynthesis and mycelial growth. Citral inhibits fungal growth by damaging oxidative phosphorylation and cell membranes through massive ROS accumulation [12,16]. Citral could be added to many ﬁnished products. The maximum acceptable concentrations of citral in ﬁnished products are reviewed in the literature. For example, the maximum acceptable concentration of citral in hand-cream products is about 0.15%; in products applied to the hair with some hand contact is about 0.2%; and in products with body and hand exposure but which are primarily rinsed-off is about 1.2% [18]. Keratinocyte are a primary cell type in the epidermis that forms an essential barrier against invading microorganisms. Keratinocytes are also involved in innate immune de- fense mechanisms. Previous studies have shown that keratinocytes induces the production of pro-inﬂammatory cytokines by co-culturing with Malassezia yeasts [19,20]. Keratinocytes also produce different antimicrobial peptides, such as the defensins family, contributing to host defense against microorganisms. Cymbopogon citratus possesses various pharmacological activities, but little is known about the immunomodulating effects on keratinocytes. Although several studies have demonstrated that citral possesses antifungal, antimicrobial, and anti-inﬂammatory ac- tivities [16,17], no studies have investigated its antifungal activity and action mechanism against M. furfur. Citral’s yeast apoptotic process has been reported in common disease- causing yeasts such as Candida albican [3,21–23], but not in Malassezia spp. Therefore, this study aimed to investigate the inhibitory activity of citral against M. furfur and its im- munomodulatory effect on keratinocytes in vitro. 2. Materials and Methods 2.1. Materials Citral (Sigma-Aldrich, St. Louis, MO, USA) with 96% purity was dissolved in dimethyl sulfoxide (DMSO) to prepare the stock solution just before performing the assays. The antifungal drug KTZ (Sigma-Aldrich) stock solution was prepared using DMSO as the solvent and stored at 20 C. The ﬁnal concentration of DMSO in every assay was 1%. 2.2. Microorganism and Cultivation M. furfur BCRC 22,243 was purchased from the Bioresource Collection and Research Center (BCRC; Taiwan). To prepare budding yeast suspensions, the strain was grown in modiﬁed Dixon (mDixon) medium (3.6% malt extract, 2.0% desiccated ox bile, 0.6% peptone, 1.0% tween 40, and 0.2% oleic acid, pH 6.0) and incubated for three days at 30 C. For the human keratinocyte invasion experiment, M. furfur was cultured on agar plates of modiﬁed Leeming and Notman (mLNA) agar (1% peptone, 1% glucose, 0.2% yeast extract, 0.8% desiccated ox bile, 1% glycerol, 0.05% glycerol monostearate, 0.5% tween 60, 2% olive oil, and 1.5% agar, pH 6.0) for three days at 30 C [24]. 2.3. Antifungal Susceptibility Tests The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of citral to M. furfur were determined using the microdilution broth method. Serial Processes 2022, 10, 802 3 of 13 two-fold dilutions of citral were made in 24-well microtiter plates to obtain concentrations of 1.0 to 1000 g/mL. M. furfur was suspended in mDixon medium to the ﬁnal density of 5 10 CFU/mL. Each well was inoculated with 0.5 mL of the inoculum suspension and incubated at 30 C for 48 h. KTZ was utilized as the control drug. The MIC was deﬁned as the lowest drug concentration that would inhibit the visible growth of a microorganism after incubation. MFC was determined as the lowest drug concentration that killed >99.9% of the initial inoculum. To determine MFC, 50 L from each well showing no growth was spread on the mDixon plates and incubated at 30 C for 72 h [25]. In the time–killing assay, yeasts were treated with different concentrations of citral. After incubation at various time points, the cells were plated out on mDixon plates for viable counts. 2.4. Morphological Analysis M. furfur cells were stained with lactophenol cotton blue (LPCB) staining after being treated with different concentrations of citral for 48 h at 30 C. The proportions of yeast to mycelial conversion of M. furfur treated with various concentrations of citral were determined using a light microscopy at 1000 magniﬁcation [7]. 2.5. Analysis of Apoptosis Markers 2.5.1. Phosphatidylserine Externalization For early-stage apoptotic marker analysis, phosphatidylserine externalization was determined using the Annexin V-FITC apoptosis detection kit (BD Pharmingen, San Jose, CA, USA). M. furfur yeast cells (1 10 CFU/mL) were harvested by centrifugation, and washed and digested with lysing enzyme (20 mg/mL) and lyticase (50 U/L) in 0.1 M potassium phosphate buffer (PPB; pH 6.0) containing 1 M sorbitol for 2 h at 30 C. Protoplasts of M. furfur were incubated with citral or H O for 2 h at 30 C, washed and 2 2 resuspended in annexin binding buffer. Cells were incubated with 5 L/mL of annexin V-FITC and propidium iodide (PI) for 20 min. Flow cytometry was performed with a FACSCalibur ﬂow cytometer (Becton Dickinson, San Jose, CA, USA) [26]. 2.5.2. TUNEL Assay DNA fragmentation was analyzed by the terminal deoxynucleotidyl transferase dUTP TM nick-end labeling (TUNEL) assay using APO-DIRECT kit (BD Pharmingen) to observe late stage of apoptosis. Cells (1 10 CFU/mL) were treated with citral for 24 h at 30 C, then ﬁxed and protoplasted, labeled with DNA labeling solution, and stained with PI. Cells were then analyzed with a FACSCalibur ﬂow cytometer [27]. 2.5.3. Metacaspase Activation Activated metacaspases in M. furfur were measured using the CaspACE FITC-VAD- FMK In Situ Marker (Promega, Madison WI). Cells (1 10 CFU/mL) were treated with citral or H O for 3 h at 30 C. The cells were washed in PBS, suspended in a staining 2 2 solution containing 10 M FITC-VAD-FMK, incubated for 20 min at room temperature in the dark, and analyzed with a FACSCalibur ﬂow cytometer [28]. 2.6. Cell Culture A human keratinocyte (HaCaT) cell line was cultured as monolayers in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 g/mL streptomycin at 37 C with 5% CO . The medium was changed every 2 days. 2.7. MTT Assay The cytotoxic effect of citral on HaCaT cells was determined using MTT assay. HaCaT cells (5 10 cells/mL) were treated with 25–800 g/mL citral at 37 C for 48 and 72 h. The medium was removed and the cells were washed once with PBS; DMEM containing Processes 2022, 10, 802 4 of 13 0.5 mg/mL MTT was added, and the cells were incubated at 37 C for 4 h. The absorbance was measured at 570 nm. The data are expressed as the percentage of viable cells compared to the 1% DMSO-treated control [2]. 2.8. Treatment of HaCaT Cells with M. furfur in the Presence or Absence of Citral HaCaT cells were infected with M. furfur at a ratio of 1:20 or 1:30 (HaCaT cells:yeasts), and treated with or without citral for 24 and 48 h. After treatment, cells were washed with PBS and stained with May–Grunwald and Giemsa stain, then examined under a light microscope. The percentage invasiveness was determined by counting the HaCaT cells that the M. furfur yeast had penetrated. The infected cells were treated with or without DMEM containing 0.25 g/mL KTZ for 4 h at 37 C. After this period, cells were scraped and diluted in PBS and plated on mLNA plates. The plates were then incubated for 72 h at 30 C, and colonies were counted (M1 cfu/mL). Yeast cells treated with KTZ were also counted (M0 cfu/mL). The adhesion percentage was then calculated as % adhesion = (M0 M1/M0) *100 [29]. 2.9. ELISA Analysis For measuring the secreted IL-6, IL-10, TNF-, TGF- , and TSLP, cell culture super- natants were collected and tested using the human cytokine ELISA kit (Invitrogen, CA, USA; TSLP from eBioscience) according to the manufacturer ’s instruction. 2.10. RNA Extraction and RT-PCR Analysis Total RNAs were isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and RNeasy Mini Kit (Qiagen, Germantown, MD, USA) from the HaCaT cells treated and not treated with M. furfur, then cDNA was synthesized with the random hexamer primers using the RevertAid H Minus First Stand cDNA Synthesis Kit (Thermo Scientiﬁc, Madison, WI, USA). Real-time RT-PCR (ABI Step One real-time PCR system, Foster City, CA, USA) was used to analyze the mRNA expression of the target genes with the Maxima SYBR Green qPCR master mix kit (Fermentas, Waltham, MA, USA) according to the manufacturer ’s protocol. Table 1 summarizes the primer sets information and reaction conditions. Table 1. Primer sets for real-time RT-PCR. Gene Sense and Anti-Sense Sequence Size (bp) 0 0 5 -TGTCTTGTGACCGCAATGGT-3 TLR2 101 0 0 5 -TGTTGGACAGGTCAAGGCTTT-3 0 0 5 -TAGCAATCGGCCACATTGCC-3 TSLP 145 0 0 5 -CTGAGTTTCCGAATAGCCTG-3 0 0 5 -ATCAGCCATGAGGGTCTTGT-3 HBD-2 172 0 0 5 -GAGACCACAGGTGCCAATTT-3 0 0 5 -TGAACGGGAAGCTCACTGG-3 GAPDH 307 5 -TCCACCACCCTGTTGCTGTA-3 TLR2, Toll-like receptor 2 [2]; TSLP, thymic stromal lymphopoietin [30]; HBD-2, human beta-defensin 2 [31]; GAPDH, glyceraldehyde-3-phosphate dehydrogenase [32]. 2.11. Statistical Analysis Each experiment was performed at least in triplicate. Results were analyzed for statistical signiﬁcance by unpaired Student’s t-test using SigmaPlot v14. A p-value < 0.05 was considered statistically signiﬁcant. 3. Results 3.1. Antifungal Activity of Citral against M. furfur The MIC and MFC of citral against M. furfur were analyzed in triplicate using the microdilution method to determine the fungicidal activities of the citral. KTZ was used as a positive control. The MIC and MFC of citral were 200 g/mL and 300 g/mL, and Processes 2022, 10, x FOR PEER REVIEW 5 of 14 Processes 2022, 10, x FOR PEER REVIEW 5 of 14 Each experiment was performed at least in triplicate. Results were analyzed for sta- Each experiment was performed at least in triplicate. Results were analyzed for sta- tistical significance by unpaired Student’s t-test using SigmaPlot v14. A p-value < 0.05 was tistical significance by unpaired Student’s t-test using SigmaPlot v14. A p-value < 0.05 was considered statistically significant. considered statistically significant. 3. Results 3. Results 3.1. Antifungal Activity of Citral against M. furfur 3.1. Antifungal Activity of Citral against M. furfur The MIC and MFC of citral against M. furfur were analyzed in triplicate using the The MIC and MFC of citral against M. furfur were analyzed in triplicate using the microdilution method to determine the fungicidal activities of the citral. KTZ was used as Processes 2022, 10, 802 5 of 13 microdilution method to determine the fungicidal activities of the citral. KTZ was used as a positive control. The MIC and MFC of citral were 200 μg/mL and 300 μg/mL, and KTZ a positive control. The MIC and MFC of citral were 200 μg/mL and 300 μg/mL, and KTZ were 0.13 μg/mL and 0.25 μg/mL, respectively (Table 2). The MIC and MFC of citral were 0.13 μg/mL and 0.25 μg/mL, respectively (Table 2). The MIC and MFC of citral against M. furfur were higher than those of known antifungal agents such as KTZ. The KTZ were 0.13 g/mL and 0.25 g/mL, respectively (Table 2). The MIC and MFC of citral against M. furfur were higher than those of known antifungal agents such as KTZ. The time–killing curves showed that the fungicidal activity of citral against M. furfur de- against M. furfur were higher than those of known antifungal agents such as KTZ. The time–killing curves showed that the fungicidal activity of citral against M. furfur de- pended on its concentration (Figure 1). Citrate exhibited rapid killing during the first 2–4 time–killing curves showed that the fungicidal activity of citral against M. furfur depended pended on its concentration (Figure 1). Citrate exhibited rapid killing during the first 2–4 h of the assay above MFC. Citral treatment also significantly increased the proportions of on its concentration (Figure 1). Citrate exhibited rapid killing during the ﬁrst 2–4 h of the h of the assay above MFC. Citral treatment also significantly increased the proportions of yeast cells to mycelial forms, by approximately 2.6-fold (Figure 2). assay above MFC. Citral treatment also signiﬁcantly increased the proportions of yeast cells yeast cells to mycelial forms, by approximately 2.6-fold (Figure 2). to mycelial forms, by approximately 2.6-fold (Figure 2). Table 2. The antifungal activity of citral and ketoconazole (KTZ) against M. furfur. Table 2. The antifungal activity of citral and ketoconazole (KTZ) against M. furfur. Table 2. The antifungal activity of citral and ketoconazole (KTZ) against M. furfur. MIC (μg/mL) MFC (μg/mL) MIC (μg/mL) MFC (μg/mL) MIC (g/mL) MFC (g/mL) Citral 200 ± 15 300 ± 25 Citral 200 ± 15 300 ± 25 Citral 200 15 300 25 Ket Ketoconazole oconazole 0.1 0.13 3 ± 0.05 0.05 0.20.25 5 ± 0.09 0.09 Ketoconazole 0.13 ± 0.05 0.25 ± 0.09 All results are the means of three determinations. All results are the means of three determinations. All results are the means of three determinations. Figure 1. Time–killing curves of the citral against Malassezia furfur. M. furfur was treated with dif- Figure 1. Time–killing curves of the citral against Malassezia furfur. M. furfur was treated with Figure 1. Time–killing curves of the citral against Malassezia furfur. M. furfur was treated with dif- ferent concentrations of citral in the mDixon medium. After 1, 2, 3, and 4 h, the yeasts were spread different concentrations of citral in the mDixon medium. After 1, 2, 3, and 4 h, the yeasts were spread ferent concentrations of citral in the mDixon medium. After 1, 2, 3, and 4 h, the yeasts were spread on the mDixon plates and incubated for 72 h at 30 °C. The colony-forming units were counted and on the mDixon plates and incubated for 72 h at 30 C. The colony-forming units were counted and on the mDixon plates and incubated for 72 h at 30 °C. The colony-forming units were counted and compared to control plates. compared to control plates. compared to control plates. Figure 2. Citral increased the proportions of yeast cells to mycelial forms. M. furfur was treated with Figure 2. Citral increased the proportions of yeast cells to mycelial forms. M. furfur was treated with Figure 2. Citral increased the proportions of yeast cells to mycelial forms. M. furfur was treated with different concentrations of citral for 48 h at 30 °C. Yeast cells were stained with the LPCB method different concentrations of citral for 48 h at 30 °C. Yeast cells were stained with the LPCB method different concentrations of citral for 48 h at 30 C. Yeast cells were stained with the LPCB method and and examined at 1000× magnification using a light microscope. and examined at 1000× magnification using a light microscope. examined at 1000 magniﬁcation using a light microscope. 3.2. Citral Induced Apoptosis in M. furfur 3.2.1. Phosphatidylserine Externalization In combination with membrane-impermeable dye, PI, Annexin V-FITC stain, which 2+ binds to phosphatidylserine with high afﬁnity in the presence of Ca , was used to deter- mine the citral-induced apoptosis in M. furfur. As shown in Figure 3, citral-treated M. furfur cells showed similar phosphatidylserine externalizations to H O -treated M. furfur cells in 2 2 which H O was an inducer of apoptosis in the yeast cells. 2 2 Processes 2022, 10, x FOR PEER REVIEW 6 of 14 3.2. Citral Induced Apoptosis in M. furfur 3.2.1. Phosphatidylserine Externalization In combination with membrane-impermeable dye, PI, Annexin V-FITC stain, which 2+ binds to phosphatidylserine with high affinity in the presence of Ca , was used to deter- mine the citral-induced apoptosis in M. furfur. As shown in Figure 3, citral-treated M. fur- Processes 2022, 10, 802 6 of 13 fur cells showed similar phosphatidylserine externalizations to H2O2-treated M. furfur cells in which H2O2 was an inducer of apoptosis in the yeast cells. Figure 3. Effect of citral on externalization of phosphatidylserine at the cytoplasmic membrane. Figure 3. Effect of citral on externalization of phosphatidylserine at the cytoplasmic membrane. Phosphatidylserine externalizations were determined by Annexin V-FITC and PI staining in citral- Phosphatidylserine externalizations were determined by Annexin V-FITC and PI staining in citral-or or H2O2-treated M. furfur cells using a flow cytometer. (A) Control, (B) 10 mM H2O2 as positive con- H O -treated M. furfur cells using a ﬂow cytometer. (A) Control, (B) 10 mM H O as positive control, 2 2 2 2 trol, (C) 300 μg/mL citral, and (D) 600 μg/mL citral. (C) 300 g/mL citral, and (D) 600 g/mL citral. 3.2.2. DNA Fragmentation 3.2.2. DNA Fragmentation DNA fragmentation is one of the late apoptotic phenotypes. To investigate whether DNA fragmentation is one of the late apoptotic phenotypes. To investigate whether citral induces the late stage of apoptosis in M. furfur, we evaluated DNA fragmentation citral induces the late stage of apoptosis in M. furfur, we evaluated DNA fragmentation using TUNEL assay and flow cytometry. TUNEL assay is a standard method for detecting using TUNEL assay and ﬂow cytometry. TUNEL assay is a standard method for detecting apoptotic DNA cleavage in individual nuclei by labeling the fluorescent dUTP at the 3′- apoptotic DNA cleavage in individual nuclei by labeling the ﬂuorescent dUTP at the 3 -OH Processes 2022, 10, x FOR PEER REVIEW 7 of 14 OH ends of DNA. We found that M. furfur cells exposed to citral and H2O2 showed in- ends of DNA. We found that M. furfur cells exposed to citral and H O showed increased 2 2 creased fluorescence intensity compared to untreated cells (Figure 4). ﬂuorescence intensity compared to untreated cells (Figure 4). Figure 4. Effect of citral-induced DNA fragmentation in M. furfur cells. Cells were treated with citral Figure 4. Effect of citral-induced DNA fragmentation in M. furfur cells. Cells were treated with or H2O2 for (A) 3 h and (B) 24 h. Cells were stained with TUNEL staining and analyzed using flow citral or H O for (A) 3 h and (B) 24 h. Cells were stained with TUNEL staining and analyzed using 2 2 cytometry. ﬂow cytometry. 3.2.3. Metacaspase Activation The activation of metacaspase plays a vital role in apoptosis. Citral-treated M. furfur cells were incubated with FITC-labeled VAD-FMK to monitor the metacaspase activation. The FITC-labeled caspase inhibitor VAD-FMK, a cell-permeable fluorescent maker, binds specifically to the active center of metazoan caspases in apoptotic cell. As shown in Figure 5, cells treated with citral showed increased fluorescence intensity, consistent with the positive control cells treated with H2O2. These results suggest that citral induces the bio- chemical feature of apoptosis in M. furfur cells, including membrane depolarization, DNA fragmentation, and metacaspase activation. Figure 5. Effect of citral on the activity of metacaspase in M. furfur cells. Cells were stained with FITC-VAD-FMK, and analyzed using a flow cytometer. (A) 10 mM H2O2, (B) 300 μg/mL citral, and (C) 600 μg/mL citral. Processes 2022, 10, x FOR PEER REVIEW 7 of 14 Figure 4. Effect of citral-induced DNA fragmentation in M. furfur cells. Cells were treated with citral Processes 2022, 10, 802 7 of 13 or H2O2 for (A) 3 h and (B) 24 h. Cells were stained with TUNEL staining and analyzed using flow cytometry. 3.2.3. Metacaspase Activation 3.2.3. Metacaspase Activation The activation of metacaspase plays a vital role in apoptosis. Citral-treated M. furfur The activation of metacaspase plays a vital role in apoptosis. Citral-treated M. furfur cells were incubated with FITC-labeled VAD-FMK to monitor the metacaspase activation. cells were incubated with FITC-labeled VAD-FMK to monitor the metacaspase activation. The FITC-labeled caspase inhibitor VAD-FMK, a cell-permeable fluorescent maker, binds The FITC-labeled caspase inhibitor VAD-FMK, a cell-permeable ﬂuorescent maker, binds specifically to the active center of metazoan caspases in apoptotic cell. As shown in Figure speciﬁcally to the active center of metazoan caspases in apoptotic cell. As shown in 5, cells treated with citral showed increased fluorescence intensity, consistent with the Figure 5, cells treated with citral showed increased ﬂuorescence intensity, consistent with positive control cells treated with H2O2. These results suggest that citral induces the bio- the positive control cells treated with H O . These results suggest that citral induces the 2 2 chemica biochemical l feature o featur f apopt e of apoptosis osis in M. furfu in M. r cel furfur ls, incl cells, udiincl ng membran uding membr e depol ane ardepolari ization, DNA zation, fra DNA gment fragmentation, ation, and meand tacasp metacaspase ase activatiactivation. on. Processes 2022, 10, x FOR PEER REVIEW 8 of 14 Figure 5. Effect of citral on the activity of metacaspase in M. furfur cells. Cells were stained with Figure 5. Effect of citral on the activity of metacaspase in M. furfur cells. Cells were stained with FITC-VAD-FMK, and analyzed using a ﬂow cytometer. (A) 10 mM H O (B) 300 g/mL citral, and 2 2, FITC-VAD-FMK, and analyzed using a flow cytometer. (A) 10 mM H2O2, (B) 300 μg/mL citral, and (C) 600 g/mL citral. (C) 600 μg/mL citral. 3.3. Citral Can Decrease the Adhesiveness and Invasiveness of M. furfur to Human 3.3. Citral Can Decrease the Adhesiveness and Invasiveness of M. furfur to Human Keratinocytes Keratinocytes at Sub-MIC at Sub-MIC 3.3.1. The Cytotoxic Effect of Citral on HaCaT Cells 3.3.1. The Cytotoxic Effect of Citral on HaCaT Cells For determine the cytotoxic effects in HaCaT cells, cell viabilities were measured us- For determine the cytotoxic effects in HaCaT cells, cell viabilities were measured ing an MTT assay. After treatment with different concentrations of citral, the viability per- using an MTT assay. After treatment with different concentrations of citral, the viability centages of HaCaT cells were >98% (Figure 6). These results demonstrate that citral has no percentages of HaCaT cells were >98% (Figure 6). These results demonstrate that citral has cyt no o cytotoxic toxic effe ef ctfect in H in aHaCaT CaT cellcells s at tat he ant the antifungal ifungal conc concentrations. entrations. Figure 6. Cell viability of HaCaT cells to citral. HaCaT cells were treated with different concentrations Figure 6. Cell viability of HaCaT cells to citral. HaCaT cells were treated with different concentra- of citral for (A) 48 h and (B) 72 h. An MTT assay was used to determine the cell viabilities. tions of citral for (A) 48 h and (B) 72 h. An MTT assay was used to determine the cell viabilities. 3.3.2. The Inhibitory Effect of Citral on M. furfur Adhesion to HaCaT Cells 3.3.2. The Inhibitory Effect of Citral on M. furfur Adhesion to HaCaT Cells For determining whether citral could interfere with M. furfur invasion, the HaCaT For determining whether citral could interfere with M. furfur invasion, the HaCaT cells were treated with different concentrations of citral and infected with M. furfur (1:20 or cells were treated with different concentrations of citral and infected with M. furfur (1:20 1:30, cells:yeasts) for 24 h. We can found that at low concentrations (25, 50 g/mL), citral or 1:30, cells:yeasts) for 24 h. We can found that at low concentrations (25, 50 μg/mL), citral can inhibit the adhesion of M. furfur to HaCaT cells and the inhibition was more apparent can inhibit the adhesion of M. furfur to HaCaT cells and the inhibition was more apparent at higher concentrations (100, 200 g/mL) compared with the control (Figure 7). at higher concentrations (100, 200 μg/mL) compared with the control (Figure 7). Figure 7. The effect of citral against M. furfur adherence on HaCaT cells. HaCaT cells were cultured with citral and M. furfur at a ratio of 1:20 or 1:30 (HaCaT cells:yeasts). After 24 h, the infected cells were treated with or without DMEM containing 0.25 μg/mL ktz for 4 h. The adhesion percentage was then calculated as % adhesion = (M0 − M1/M0) *·100. (M1,CFU of treated with various citral concentrations; M0, CFU of treated with ktz). Adherence differences were determined using the unpaired Student’s t-test (* p < 0.05). Processes 2022, 10, x FOR PEER REVIEW 8 of 14 3.3. Citral Can Decrease the Adhesiveness and Invasiveness of M. furfur to Human Keratinocytes at Sub-MIC 3.3.1. The Cytotoxic Effect of Citral on HaCaT Cells For determine the cytotoxic effects in HaCaT cells, cell viabilities were measured us- ing an MTT assay. After treatment with different concentrations of citral, the viability per- centages of HaCaT cells were >98% (Figure 6). These results demonstrate that citral has no cytotoxic effect in HaCaT cells at the antifungal concentrations. Figure 6. Cell viability of HaCaT cells to citral. HaCaT cells were treated with different concentra- tions of citral for (A) 48 h and (B) 72 h. An MTT assay was used to determine the cell viabilities. 3.3.2. The Inhibitory Effect of Citral on M. furfur Adhesion to HaCaT Cells For determining whether citral could interfere with M. furfur invasion, the HaCaT cells were treated with different concentrations of citral and infected with M. furfur (1:20 or 1:30, cells:yeasts) for 24 h. We can found that at low concentrations (25, 50 μg/mL), citral Processes 2022, 10, 802 8 of 13 can inhibit the adhesion of M. furfur to HaCaT cells and the inhibition was more apparent at higher concentrations (100, 200 μg/mL) compared with the control (Figure 7). Figure 7. The effect of citral against M. furfur adherence on HaCaT cells. HaCaT cells were cultured Figure 7. The effect of citral against M. furfur adherence on HaCaT cells. HaCaT cells were cultured with citral and M. furfur at a ratio of 1:20 or 1:30 (HaCaT cells:yeasts). After 24 h, the infected cells with citral and M. furfur at a ratio of 1:20 or 1:30 (HaCaT cells:yeasts). After 24 h, the infected cells were treated with or without DMEM containing 0.25 μg/mL ktz for 4 h. The adhesion percentage were treated with or without DMEM containing 0.25 g/mL ktz for 4 h. The adhesion percentage was then calculated as % adhesion = (M0 − M1/M0) *·100. (M1,CFU of treated with various citral Processes 2022, 10, x FOR PEER REVIEW was then calculated as % adhesion = (M0 M1/M0) *100. (M1,CFU of treated with various 9 of citral 14 concentrations; M0, CFU of treated with ktz). Adherence differences were determined using the concentrations; M0, CFU of treated with ktz). Adherence differences were determined using the unpaired Student’s t-test (* p < 0.05). unpaired Student’s t-test (* p < 0.05). 3.3.3. The Inhibitory Effect of Citral on M. furfur Invasiveness into HaCaT Cells 3.3.3. The Inhibitory Effect of Citral on M. furfur Invasiveness into HaCaT Cells The invasion of M. furfur to HaCaT cells was monitored using May−Grunwald and The invasion of M. furfur to HaCaT cells was monitored using MayGrunwald and Giemsa staining. Some HaCaT cells showed yeast engulfment in the negative-control Giemsa staining. Some HaCaT cells showed yeast engulfment in the negative-control group group and low-concentration citral-treatment groups (Figure 8A). Cell invasiveness of and low-concentration citral-treatment groups (Figure 8A). Cell invasiveness of 15% and 15% and 30% were determined at HaCaT cells to yeasts ratios of 1:20 and 1:30 (Figure 8B). 30% were determined at HaCaT cells to yeasts ratios of 1:20 and 1:30 (Figure 8B). Like the Like the adhesion, citral reduced the invasion of M. furfur to HaCaT cells (Figure 8C). adhesion, citral reduced the invasion of M. furfur to HaCaT cells (Figure 8C). Figure 8. The effect of citral against M. furfur invasion on HaCaT cells. HaCaT cells treated with Figure 8. The effect of citral against M. furfur invasion on HaCaT cells. HaCaT cells treated with citral and M. furfur at a ratio of 1:20 and 1:30 (HaCaT cells:yeasts) for 24 and 48 h. (A) HaCaT cells citral and M. furfur at a ratio of 1:20 and 1:30 (HaCaT cells:yeasts) for 24 and 48 h. (A) HaCaT cells were stained with the May–Grunwald and Giemsa method after incubation with yeast. Some cells were stained with the May–Grunwald and Giemsa method after incubation with yeast. Some cells show yeast engulfment. (B, C) Yeasts were taken up into HaCaT cells after being treated different show yeast engulfment. (B,C) Yeasts were taken up into HaCaT cells after being treated different concentrations of citral for 24 and 48 h. The percentage of invasiveness was determined by counting concentrations of citral for 24 and 48 h. The percentage of invasiveness was determined by counting the HaCaT cells that the M. furfur yeast had penetrated. The negative control group (cells:yeasts the HaCaT cells that the M. furfur yeast had penetrated. The negative control group (cells:yeasts ratio ratio of 1:30) was set as 100%. Differences in adherence were determined using the unpaired Stu- of 1:30) was set as 100%. Differences in adherence were determined using the unpaired Student’s dent’s t-test (* p < 0.05). t-test (* p < 0.05). 3.4. Citral Can Modulate the Immune Response of HUMAN keratinocytes and Interfere with M. furfur Infection 3.4.1. TLR2, HBD-2, and TSLP Gene Expression We next examined the effect of citral on the mRNA expression of TLR2, HBD-2, and TSLP by real-time RT-PCR in the M. furfur-infected HaCaT cells. HaCaT cells were treated with M. furfur (cells:yeasts of 1:30) at different citral sub-MICs. The TLR2 mRNA expres- sion was 30-fold higher in M. furfur-treated HaCaT cells at 8 h treatment. In contrast, when cells were cotreated with M. furfur and citral, the increased TLR2 mRNA expression was significantly downregulated by citral (Figure 9A), and the TLR2 mRNA expression down- regulations were also found after 24 h treatment (Figure 9B). Moreover, both citral and M. furfur induced HBD-2 production, with the strongest effect occurring in M. furfur-infected cells after 24 h treatment (Figure 10). Finally, we tested the effect of citral on TLSP mRNA expression. The TSLP mRNA expression increased in the M. furfur-infected cells, and citral had a more apparent effect on TSLP transcript after 24 h treatment (Figure 11). These re- sults demonstrate that citral efficiently inhibits the M. furfur-induced TLR2 mRNA expres- sion and enhances HBD-2 and TSLP mRNA expression in HaCaT cells. Processes 2022, 10, 802 9 of 13 3.4. Citral Can Modulate the Immune Response of HUMAN keratinocytes and Interfere with M. furfur Infection 3.4.1. TLR2, HBD-2, and TSLP Gene Expression We next examined the effect of citral on the mRNA expression of TLR2, HBD-2, and TSLP by real-time RT-PCR in the M. furfur-infected HaCaT cells. HaCaT cells were treated with M. furfur (cells:yeasts of 1:30) at different citral sub-MICs. The TLR2 mRNA expression was 30-fold higher in M. furfur-treated HaCaT cells at 8 h treatment. In contrast, when cells were cotreated with M. furfur and citral, the increased TLR2 mRNA expression was signiﬁcantly downregulated by citral (Figure 9A), and the TLR2 mRNA expression downregulations were also found after 24 h treatment (Figure 9B). Moreover, both citral and M. furfur induced HBD-2 production, with the strongest effect occurring in M. furfur- infected cells after 24 h treatment (Figure 10). Finally, we tested the effect of citral on TLSP mRNA expression. The TSLP mRNA expression increased in the M. furfur-infected cells, and citral had a more apparent effect on TSLP transcript after 24 h treatment (Figure 11). Processes 2022, 10, x FOR PEER REVIEW 10 of 14 These results demonstrate that citral efﬁciently inhibits the M. furfur-induced TLR2 mRNA Processes 2022, 10, x FOR PEER REVIEW 10 of 14 expression and enhances HBD-2 and TSLP mRNA expression in HaCaT cells. Figure 9. The gene expression of TLR2 in citral against M. furfur-infected HaCaT cells. HaCaT cells Figure 9. The gene expression of TLR2 in citral against M. furfur-infected HaCaT cells. HaCaT cells Figure 9. The gene expression of TLR2 in citral against M. furfur-infected HaCaT cells. HaCaT cells were cotreated with citral and M. furfur at a cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. Cells were cotreated with citral and M. furfur at a cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. Cells were cotreated with citral and M. furfur at a cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. Cells were scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine the TLR2 were scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine the TLR2 were scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine the TLR2 mRNA expression. All values are expressed as mean ± SD. Differences in the expression of TLR2 mRNA expression. All values are expressed as mean SD. Differences in the expression of TLR2 mRNA expression. All values are expressed as mean ± SD. Differences in the expression of TLR2 were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with non-treat- were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with non-treatment were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with non-treat- ment control, # means comparison with M. furfur-infected cells.). ment control, # means comparison with M. furfur-infected cells.). control, # means comparison with M. furfur-infected cells.). Figure 10. The gene expression of HBD-2 in citral against M. furfur-infected HaCaT cells. HaCaT Figure 10. The gene expression of HBD-2 in citral against M. furfur-infected HaCaT cells. HaCaT Figure 10. The gene expression of HBD-2 in citral against M. furfur-infected HaCaT cells. HaCaT cells were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. cells were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. cells were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. Cells were scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine the Cells were scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine the HBD-2 mRNA Cells were scrap-harvested expression. Aland l values quantitatively are expressed as mean ± SD analyzed using real-time . Differences in the RT-PCR to expression determine of the HBD-2 mRNA expression. All values are expressed as mean ± SD. Differences in the expression of HBD-2 were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with non- HBD-2 mRNA expression. All values are expressed as mean SD. Differences in the expression HBD-2 were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with non- treatment control, # means comparison with M. furfur-infected cells.). of HBD-2 were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with treatment control, # means comparison with M. furfur-infected cells.). non-treatment control, # means comparison with M. furfur-infected cells.). Figure 11. The gene expression of TSLP in citral against M. furfur-infected HaCaT cells. HaCaT cells Figure 11. The gene expression of TSLP in citral against M. furfur-infected HaCaT cells. HaCaT cells were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for 24 h. Cells were scrap- were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for 24 h. Cells were scrap- harvested and quantitatively analyzed using real-time RT-PCR to determine TSLP mRNA expres- harvested and quantitatively analyzed using real-time RT-PCR to determine TSLP mRNA expres- sion. All values are expressed as mean ± SD. Differences in TSLP expression were determined using sion. All values are expressed as mean ± SD. Differences in TSLP expression were determined using Processes 2022, 10, x FOR PEER REVIEW 10 of 14 Figure 9. The gene expression of TLR2 in citral against M. furfur-infected HaCaT cells. HaCaT cells were cotreated with citral and M. furfur at a cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. Cells were scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine the TLR2 mRNA expression. All values are expressed as mean ± SD. Differences in the expression of TLR2 were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with non-treat- ment control, # means comparison with M. furfur-infected cells.). Figure 10. The gene expression of HBD-2 in citral against M. furfur-infected HaCaT cells. HaCaT cells were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for (A) 8 h and (B) 24 h. Cells were scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine the HBD-2 mRNA expression. All values are expressed as mean ± SD. Differences in the expression of Processes 2022, 10, 802 10 of 13 HBD-2 were determined using unpaired Student’s t-test (* p < 0.05). (* means comparison with non- treatment control, # means comparison with M. furfur-infected cells.). Processes 2022, 10, x FOR PEER REVIEW 11 of 14 Figure 11. The gene expression of TSLP in citral against M. furfur-infected HaCaT cells. HaCaT cells Figure 11. The gene expression of TSLP in citral against M. furfur-infected HaCaT cells. HaCaT were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for 24 h. Cells were scrap- cells were cotreated with citral and M. furfur at cells to yeasts ratio of 1:30 for 24 h. Cells were harvested and quantitatively analyzed using real-time RT-PCR to determine TSLP mRNA expres- scrap-harvested and quantitatively analyzed using real-time RT-PCR to determine TSLP mRNA unpaired Student’s t-test (* p < 0.05). (* means comparison with non-treatment control, # means sion. All values are expressed as mean ± SD. Differences in TSLP expression were determined using expression. All values are expressed as mean SD. Differences in TSLP expression were determined comparison with M. furfur-infected cells.). using unpaired Student’s t-test (* p < 0.05). (* means comparison with non-treatment control, # means comparison with M. furfur-infected cells.). 3.4.2. Cytokine Expression Assay 3.4.2. Cytokine Expression Assay To determine how citral interferes with M. furfur, we examined the expressions of proinflammatory cytokines IL-6 and TNF-α, and anti-inflammatory cytokines IL-10 and To determine how citral interferes with M. furfur, we examined the expressions of TGF-β using ELISA. IL-6 levels increased in M. furfur-infected HaCaT cells after 24 h of proinﬂammatory cytokines IL-6 and TNF-, and anti-inﬂammatory cytokines IL-10 and trea TGF- tment. In contra using ELISA. st, IL-6 a signi levels fica incr nt decrea eased s in e in M. IL-6 furfur level w -infected as observed when t HaCaT cells afterhe cells 24 h of were trea treatment. ted In wi contrast, th citral ( aFsigniﬁcant igure 12). The resu decreaselts cle in IL-6 arly demonstr level was observed ated that c when itral the inhibits cells the proi were treated nflamwith mtory cytokine citral (FigurIL-6 expre e 12). The ssion results ind clearly uced by demonstrated M. furfur in that HaC citral aT cinhibits ells. How- the ever, expre proinﬂammtory ssion d cytokine ifference IL-6 s in TNF expression -α, IL-1 induced 0, and T by GF- M.β were n furfur inoHaCaT t detectable cells. (dat However a not , shown). expression differences in TNF-, IL-10, and TGF- were not detectable (data not shown). Figure 12. IL-6 expression in citral against M. furfur-infected HaCaT cells. The secretion of IL-6 in- Figure 12. IL-6 expression in citral against M. furfur-infected HaCaT cells. The secretion of IL-6 duced by the M. furfur-infected HaCaT cells was measured using ELISA. Cell supernatants were induced by the M. furfur-infected HaCaT cells was measured using ELISA. Cell supernatants were collected at 24 h after infection with M. furfur in the presence or absence of citral. The negative con- collected at 24 h after infection with M. furfur in the presence or absence of citral. The negative trol was the non-treated cells. The results are means of triplicate experiments. All values are ex- control was the non-treated cells. The results are means of triplicate experiments. All values are pressed as mean ± SD. Differences in IL-6 expression were determined using unpaired Student’s t- expressed as mean SD. Differences in IL-6 expression were determined using unpaired Student’s test (* p < 0.05). (* means comparison with non-treatment control, # means comparison with M. fur- t-test (* p < 0.05). (* means comparison with non-treatment control, # means comparison with M. fur-infected cells.). furfur-infected cells.). 4. Discussion 4. Discussion Malassezia spp. yeasts are dimorphic fungi and normal fungal skin flora in humans Malassezia spp. yeasts are dimorphic fungi and normal fungal skin ﬂora in humans and other warm-blooded animals. They can cause skin-related diseases through different and other warm-blooded animals. They can cause skin-related diseases through different mechanisms. Killing bacteria or bacterial inhibiting was the core concept of antibiotic de- velopment and an essential milestone in medicinal chemistry. Destroying critical compo- nents during bacterial growth is an efficient antibacterial strategy. Although clinical stud- ies have confirmed the azoles such as ketoconazole (KTZ) effectively treat Malassezia-re- lated skin diseases, the long-term safety of antifungal drugs remains unknown [7]. Tox- icity, low efficacy, and drug resistance of the antifungal drugs have limited their clinical usage. Besides, they are costly when an extended treatment time is necessary and many patients leave the therapy before being cured. One way to prevent antibiotic resistance of pathogenic species is to find and use new compounds [3,21]. Both mycelial and yeast forms have been found in several diseases believed to be caused by M. furfur. Our study showed that citral has lower antifungal activity below sub- MIC but can block the yeast to mycelia transformation of M. furfur, although the mecha- nism and effect remained unclear. In the early phases of apoptosis, phosphatidylserine is translocated from the inner leaflet of the plasma membrane bilayers to the outer leaflet. Phosphatidylserine exposure generally precedes DNA fragmentation and nuclear condensation. A putative caspase (metacaspase) has been shown to be involved in apoptosis. Our results suggested that Processes 2022, 10, 802 11 of 13 mechanisms. Killing bacteria or bacterial inhibiting was the core concept of antibiotic devel- opment and an essential milestone in medicinal chemistry. Destroying critical components during bacterial growth is an efﬁcient antibacterial strategy. Although clinical studies have conﬁrmed the azoles such as ketoconazole (KTZ) effectively treat Malassezia-related skin diseases, the long-term safety of antifungal drugs remains unknown [7]. Toxicity, low efﬁ- cacy, and drug resistance of the antifungal drugs have limited their clinical usage. Besides, they are costly when an extended treatment time is necessary and many patients leave the therapy before being cured. One way to prevent antibiotic resistance of pathogenic species is to ﬁnd and use new compounds [3,21]. Both mycelial and yeast forms have been found in several diseases believed to be caused by M. furfur. Our study showed that citral has lower antifungal activity below sub-MIC but can block the yeast to mycelia transformation of M. furfur, although the mechanism and effect remained unclear. In the early phases of apoptosis, phosphatidylserine is translocated from the inner leaﬂet of the plasma membrane bilayers to the outer leaﬂet. Phosphatidylserine exposure generally precedes DNA fragmentation and nuclear condensation. A putative caspase (metacaspase) has been shown to be involved in apoptosis. Our results suggested that citral at lower concentrations induces apoptosis in M. furfur yeast cells and necrosis at higher concentrations. The yeast apoptosis process has been found in common clinical yeast such as C. albican, but remains unclear in M. furfur [7,32–34]. In the current experiment, we chose the HaCaT cell line to examine the effect of citral on M. furfur-infected cells. At ﬁrst, M. furfur cultured in the mDixon medium barely infected the HaCaT cells. Malassezia spp. has cell walls with a very thick multi-layered structure, and the lipid compositions alter when cultivated in the different medium. The lipid layer of Malassezia spp. plays an essential role in modulating proinﬂammatory cytokine production by keratinocytes [35]. Therefore, we changed the mDixon medium to mLNA medium with higher lipid content to cultivate M. furfur for cell infection experiments and found that the infection rate of mLNA-cultured M. furfur in HaCaT cells increased signiﬁcantly compared to that of mDixon-cultured M. furfur. Toll-like receptors (TLRs) are crucial players in the innate immune responses to micro- bial invaders, TLRs and -defensins are crucial elements in the innate immune response against bacteria, fungi, and viruses affecting the human skin and TLR2 is one of the main receptors on the cell surface that recognizes fungus [2]. Our study demonstrated that citral downregulates TLR2 gene expression in M. furfur-infected keratinocytes. In addition, citral also increased the expression of HBD-2 antimicrobial peptides in HaCaT cells. The antimicrobial peptides directly bind to the cell membranes of microorganisms and alter their permeability resulting in a bacteriostatic effect. Citral may stimulate HaCaT cells to secrete antimicrobial peptides, which inhibit M. furfur growth. The binding of HBD-2 may also prevent the binding of M. furfur to cells due to steric structural barriers. In addition to recognizing foreign pathogens, TLR2 also initiates host innate immunity and affects subsequent adaptive immune responses. In recent studies, Malassezia stimulates human keratinocytes to produce cytokines and inﬂammatory molecules [20,36]. We found that citral inhibits IL-6 production in M. furfur-infected HaCaT cells. A previous study has shown that citral has an anti-inﬂammatory ability on lipopolysaccharide (LPS)-induced RAW 264.7 mouse macrophages and can inhibit the inﬂammatory cytokines molecules such as IL-6 and IL-10 [37]. Because the TLR2 precedes IL-6 and IL-8 pathways, it is reasonable to speculate that citral inhibits the TLR2 expression in HaCaT cells, resulting in a decrease in IL-6 secretion. Further research is required. 5. Conclusions In conclusion, citral has antifungal activity at high concentrations and can block morphogenesis of M. furfur at low concentrations. Citral decreases the infection of M. furfur through modulation of the keratinocyte immune response. Citral modulates the M. furfur infection in HaCaT cells by inducing the secretion of HBD-2 antimicrobial peptides. In Processes 2022, 10, 802 12 of 13 addition, citral inhibits TLR2 expression and cell recognition of M. furfur. Citral also affects the expression of M. furfur surface adhesion factor, reducing the ability of M. furfur to adhere and aggregate. Our ﬁndings suggest that citral is a potential drug component to be formulated in therapeutics for M. furfur-associated human skin diseases. Author Contributions: W.-L.L. and M.-H.L. conceived and planned the experiments. Y.-T.L. and Y.-S.L. carried out the experiments. W.-L.L. and M.-H.L. took the lead in writing the manuscript. 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Processes – Multidisciplinary Digital Publishing Institute

Published: Apr 19, 2022

Keywords: Malassezia furfur; citral; yeast apoptosis; human keratinocyte; immunomodulation

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The Inhibitory Activity of Citral against Malassezia furfur

The Inhibitory Activity of Citral against Malassezia furfur

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The Inhibitory Activity of Citral against Malassezia furfur

The Inhibitory Activity of Citral against Malassezia furfur

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