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Published in final edited form as:
J Invest Dermatol. 2013 August ; 133(8): 2023–2030. doi:10.1038/jid.2013.92.

Malassezia yeasts produce a collection of exceptionally potent
activators of the Ah (dioxin) receptor detected in diseased
human skin
Prokopios Magiatis1,2,*, Periklis Pappas3, George Gaitanis4, Nikitia Mexia1, Eleni Melliou1,5,Maria Galanou3, Christophoros Vlachos3,4, Konstantina Stathopoulou1, Alexios Leandros
Skaltsounis1, Marios Marselos3, Aristea Velegraki6, Michael S. Denison2, and Ioannis D.
Bassukas4
1Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, National
and Kapodistrian University of Athens, Panepistimiopolis, Athens 15771, Greece
2Department of Environmental Toxicology, University of California, Davis 95616, USA

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3Department of Pharmacology, Medical School, University of Ioannina, Ioannina 45110, Greece
4Department of Skin and Venereal Diseases, University Hospital of Ioannina, Medical School,

University of Ioannina, Ioannina 45110, Greece
5Department of Food Science and Technology, University of California, Davis 95616, USA
6Mycology Laboratory, Department of Microbiology, Medical School, National and Kapodistrian

University of Athens, Athens 11527, Greece

Abstract

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Malassezia yeasts are commensal microorganisms which under insufficiently understoodconditions can become pathogenic. We have previously shown that specific strains isolated from
diseased human skin can preferentially produce agonists of the aryl hydrocarbon receptor (AhR),
whose activation has been linked to certain skin diseases. Investigation of skin scale extracts frompatients with Malassezia associated diseases demonstrated 10–1000 fold higher AhR activating
capacity than control skin extracts. LC/MS/MS analysis of the patients’ extracts revealed thepresence of indirubin, 6-formylindolo[3,2-b]carbazole (FICZ), indolo[3,2-b]carbazole (ICZ),
malassezin, and pityriacitrin. The same compounds were also identified in 9/12 Malassezia species
culture extracts tested, connecting their presence in skin scales with this yeast. Studying theactivity of the Malassezia culture-extracts and pure metabolites in HaCaT cells by Reverse
Transcriptase Real-Time PCR revealed significant alterations in mRNA levels of the endogenous
AhR-responsive genes Cyp1A1, Cyp1B1 and AhRR. Indirubin and FICZ activated AhR in HaCaTand human HepG2 cells with significantly higher, yet transient, potency as compared to theprototypical AhR ligand, dioxin. In loco synthesis of these highly potent AhR inducers by
Malassezia yeasts could have a significant impact on skin homeostatic mechanisms and diseasedevelopment.

*

Corresponding author: Prokopios Magiatis, Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy,
National & Kapodistrian University of Athens, Athens 157 71, Greece. Tel. +30 210 7274052, Fax. +30 210 7274594,
magiatis@pharm.uoa.gr.
Conflict of Interest
The authors state no conflict of interest.
Additional detailed descriptions are provided in Supporting information.

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Page 2Introduction
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Healthy human skin harbors a significant amount of commensal yeasts belonging to thegenus Malassezia that can become pathogenic under currently inadequately understoodconditions (Gaitanis et al, 2012; Ashbee 2007). These yeasts are implicated in thepathogenesis of skin diseases with diverse clinical presentation. Malassezia yeasts can cause
pityriasis versicolor (PV), a minimally inflammatory condition despite a concomitant heavy
fungal load, which however modifies the function of melanocytes, as evidenced by theformation of hyper- or hypopigmented plaques (Thoma et al, 2005). On the other hand,
Malassezia yeasts are also implicated in exacerbations of certain inflammatory dermatoses,
such asatopic dermatitis (AD) and seborrheic dermatitis (SD) (Gupta et al, 2004). All theaforementioned skin conditions are common and may have a significant impact on thequality of life of afflicted individuals (Sugita et al, 2010). Currently, the genus Malassezia
comprises 14 species (Gaitanis et al, 2012). The most prevalent species in humans are M.
globosa, M. restricta, M. sympodialis and M. furfur, with the first two species present on theskin of almost all individuals (Tajima et al, 2008). A major challenge in our effort tocomprehend the role of Malassezia yeasts inskin diseases is to delineate pathogenic species
or strains and to attribute virulence factors. During the last decade an array of indolic
substances synthesized in vitro by M. furfur has been identified. These substances have been
associated with the pathogenic potential of this species (Mayser & Gaitainis 2010). We havepreviously observed that M. furfur strains isolated from SD lesions (and from PV;
unpublished observation) synthesize substances as are malassezin, indole[3,2-b]carbazole(ICZ) and pityriacitrin in significantly higher quantities in vitro compared to healthy skinisolates (Gaitanis et al, 2008). Malassezin has been shown to induce apoptosis in humanmelanocytes through Arylhydrocarbon Receptor (AhR) activation, while ICZ is a known
potent ligand of this receptor. Furthermore, the existence of malassezin on human skin could
be a chemical marker of the presence of M. furfur as the former has been described to be
produced only by this species. To date the production of these bioactive indoles had beenshown only in vitro and only for M. furfur species thus causing reservation in respect to theirclinical relevance.

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AhR is an orphan, ligand-dependent nuclear receptor with multifaceted biological functions.
It has been shown to participate in the maintenance of skin homeostasis (Bock & Kohle
2009), the enhancement of wound healing (Barouti et al, 2009) and the partial mediation ofultraviolet radiation damage through the photochemical intracellular production of thepotent AhR ligand, 6-formylindolo[3,2-b]carbazole (FICZ) (Fritsche et al, 2007). Regarding
skin pathophysiology, it is well known that TCDD causes its detrimental effects throughpersistent activation of this receptor and the downstream signaling pathway. The appearanceof chloracne, a characteristic acne-like eruption, is one of the heralding signs of TCDD
intoxicationin humans (Panteleyev & Bickers 2006; Sorg et al, 2009). Furthermore,
activation of the AhR plays an important role in mediating the biological/toxicologicaleffects of a variety of environmental xenobiotics, including the Malassezia produced
indirubin and ICZ, on the immune system (Esser et al, 2009; Vlachos et al, 2012).
After documenting the preferential production of the AhR ligands ICZ and malassezin in M.
furfur SD isolates we decided to validate their in vivo existence in Malassezia associatedskin diseases (SD and PV) as compared to appropriate control samples. This was carried out
by employing the very sensitive CALUX bioassay (Denison et al, 2004) that measures theability and relative potency of a chemical or extract to activate the AhR and AhR-dependent
gene expression. The extracts from diseased skin scales revealed significantly higher AhR
activation potential in the CALUX bioassay as compared to controls. In parallel and in order
to expand the biological significance of AhR ligands synthesis by Malassezia yeasts we:1)analyzed Malassezia-culture and skin scales extracts for the existence of additional indolic

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AhR activators; 2)screened Malassezia species for the production of AhR ligands in order toestablish the wider significance of this biochemical trait in this genus; 3)assessed the effectof Malassezia culture-extracts and indolic ingredients on HaCaT keratinocytes.

Results
AhR activation by human skin scale extracts
Organic solvent skin scale extracts were prepared from 10 patients with SD (N=6) or PV(N=4) and 6 healthy volunteers and evaluated for their ability to activate AhR-dependent
gene expression in recombinant human hepatoma (HG2L7.5c1) cells containing a stablytransfected AhR-responsive luciferase reporter gene. The extracts originating from thepatients’ lesional skin scales were between 10–1000 times more potent in stimulating AhRdependent luciferase activity than those from control healthy facial skin samples (N=3) orskin samples from anatomic areas that are not expected under physiological conditions to becolonized by Malassezia yeasts (N=3)(Table 1).
Identification, isolation and quantitation of indolic compounds from Malassezia furfur
culture extracts

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Using our previously described methodology (Gaitanis et al, 2008) we reevaluated the M.
furfur culture extracts that had been included in the previous study. By chromatographic
separations we isolated and identified by NMR and mass spectroscopy three additional
indolic compounds known to be potent AhR activators, namely indirubin, FICZ andtryptanthrin. The quantitation of the more prevalent indolic AhR ligands in culture extractsof M. furfur strains was performed by HPLC/UV or LC/MS/MS using as analyticalstandards pure synthetic substances (Fig. S1). In order to rule out the likelihood of thephotochemical production of these indoles all cultures were performed in the dark. As anegative control, extracts from agar slants without Malassezia inoculum were included.
Comparison of M furfur strains isolated from healthy skin with SD isolates showed that thelatter had elevated levels of FICZ (p=0.001), indirubin (p=0.001), and tryptanthrin (p=0.005)
and pityriacitrin (p=0.001) (Table 2), in addition to the previously documented elevatedlevels of ICZ (p<0.001) and malassezin (p=0.001) (Gaitanis et al, 2008).
Identification and quantitation of selected indolic products in cultures of different
Malassezia species

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To expand the significance of indole synthesis by Malassezia species we screened Typestrains of species that are mainly isolated from human skin. At least one of these AhR
ligands was detected in 9/12 Malassezia species strains studied (Table 3). Overall, the M.
furfur pathogenic strains were the most efficient producers of these indoles (Tables 2,3).
However, with the exception of M.sympodialis and M.restricta, all other isolates fromhumans (M.furfur, M.obtusa, M.globosa, M.slooffiae, M.japonica, M.yamatoensis) inaddition to the animal isolates M.pachydermatis and M.nana, also synthesize at least someAhR ligands.
Identification of indolic Malassezia metabolites in human skin scale samples
The identification of indirubin and FICZ, along with the previously known Malassezia
metabolites malassezin, pityriacitrin and ICZ, in EtOAc extracts of human skin scales was
performed by LC/MS/MS. FICZ and ICZ were identified in 2 out of 7 patients’ samples,
pityriacitrin in 3 out of 10, malassezin in 4 out of 10, while indirubin was found in 5 out of
10. The concentration of the identified metabolites ranged between 0.2 to 260 pmol/mg oflesional skin scales extract (Table 1). None of these compounds was found in the control
skin samples.

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Relative AhR activation potency of chemically pure indolic Malassezia metabolites

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Using the human hepatoma CALUX cell bioassay, the Malassezia metabolites were
evaluated for their ability to stimulate AhR-dependent reporter gene expression at 6 hours of
incubation. All tested compounds were potent AhR activators, with ICZ and FICZ
equipotent to the prototypical AhR agonist TCDD; malassezin and tryptanthrin were 20- and200-fold less potent than TCDD, respectively. Interestingly, indirubin was 20-fold morepotent than TCDD as an activator of the AhR in the human cell line (compare EC50s of 26
pM to 523 pM, respectively)(EC50s: FICZ at 348 pM, ICZ at 600 pM, malassezin at 11 nM
and tryptanthrin at 107 nM) (Fig. 1).
Activation of AhR pathway by M. furfur culture extracts in HaCaT cells

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The significant variability of Malassezia culture extracts in their overall composition inindolic compounds with AhR activity complicates comparison of their relative potency. For
this reason, in order to compare culture extracts of different M. furfur strains, we normalized
extract dilutions for testing to 100nM ICZ. This was selected because besides being
identified in all tested strains it is one of the most active AhR-inducers. However, since ICZ
was only found in M. furfur strains, comparisons are restricted to M. furfur lesional skinisolates. Alterations in the expression of the AhR related genes (Cyp1a1, Cyp1b1, AhRR,
Aldh3a1) as well as AhR-responsive Phase II genes (Gstp1, Gstt1) (Yu et al, 2009) by theseindoles was assessed by Reverse Transcriptase Real-Time PCR. The extracts tested did not
produce any obvious signs of toxicity in HaCaT cells when used at the equivalent of 100 nM
ICZ (Fig. S2). Extracts at this ICZ equivalent concentration significantly induced expressionof two prototypical markers of the AhR signaling pathway, namely Cyp1a1 and Cyp1b1,
with the level of CYP1A1 mRNA approximately 7-fold greater than that of CYP1B1 (Fig.
2a,b). With the exception of WCH106, these extracts significantly down-regulated the levelof AhRR mRNA (Fig. 2c) and both GSTP1 and GSTT1 mRNAs (p<0.05 compared tocontrol) (Fig. S3).
Differential potency of Malassezia produced indoles to induce AhR pathway in HaCaT cells
A concentration-response protocol was applied to expose HaCaT cells to different
concentrations of ICZ, malassezin, indirubin, FICZ and tryptanthrin. A clear concentrationdependent increase in mRNA levels of the AhR related genes Cyp1A1 and Cyp1b1 wasobserved in addition to Aldh3a1 (Fig. 3 and Fig. S4). FICZ, ICZ and indirubin proved to be
more potent AhR agonists than malassezin and tryptanthrin (Fig. 3 and Fig. S4). This was
also applicable for ALDH3A1 mRNA. Interestingly, Cyp1a1 induction by FICZ, ICZ andindirubin was higher at 24h as compared to 3 and 6h, a situation that is typically reversed forthese compounds given that they are readily metabolized in intact cells (Fig. S5).

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Discussion
The data presented herein demonstrate the identification of an array of indolic metabolites
possessing potent AhR activity in skin scales of patients with SD and PV and attribute their
origin to the skin commensal and pathogen Malassezia. In addition to the previously found
ICZ and malassezin, three more compounds, indirubin, FICZ and tryptanthrin wereidentified as Malassezia metabolites. Up to now only tryptanthrin has been shown to be
produced by the yeast species Yarrowia (Candida) lipolytica (Shrenk et al, 1997) andCandida glabrata (Mayser et al, 2007). Both FICZ (Wincent et al, 2009; 2012) and indirubin(Prochazkova et al, 2011) are proposed as endogenous AhR ligands and have multiple
biological functions. Indirubin inhibits CDKs as well as the GSK-3 (Hoessel et al, 1999) andcan modulate key cellular signaling pathways like Wnt and NF-kB (Tian et al, 1999). Also
bacteria can cause the purple urine syndrome through the synthesis of indirubin (Kang et al,2011). FICZ has drawn attention as it has been implicated in the mediation of UV damage
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through AhR activation (Fritche et al, 2007). However both could also cause indirect skindamage by the local leak of free radicals after AhR induced hyperactivation of CYP
enzymes (Park et al, 1996). Presently, the identification of malassezin and pityriacitrin
exclusively in lesional skin scale extracts points to Malassezia as the source of origin.
Malassezin is uniquely produced by Malassezia species, and can be considered as achemical marker of this genus. In humans, pityriacitrin has been shown to be produced also
by Candida glabrata (Mayser et al, 2007), the colon yeast equivalent of Malassezia. Crosscontamination only of the SD and PV samples with pityriacitrin originating from Candidastrains residing in the colon is highly improbable and thus isolation or this indole from skinscales, further corroborates towards its Malassezia origin.

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Regarding the clinical significance of AhR ligand production by Malassezia yeasts we have
two important complementary findings: 1)These ligands are produced in vitro by the
majority of Malassezia species and in particular by the ubiquitous M. globosa (Tajima et al,2008) and 2)their presence was confirmed on the skin scales of PV and SD by bothanalytical and biological assays. The quantitative differences in the identified indoles fromthe skin scales could represent variations in the activity of the yet unknown biosynthetic
pathway or in loco modification by environmental factors as is the skin pH and/or solar
radiation (Fritsche et al, 2007). Thus ICZ has been proposed to be easily produced throughtransformation of malassezin (Wille et al, 2001), yet in our samples we could find either
one, or both of these two substances. We cannot attribute the production of the AhR ligands
in the skin scales solely to M. furfur as it was isolated only in 2 of our present SD patients(data not shown). Probably, as shown from our results, additional Malassezia species, as is
M. globosa (Tajima et al, 2008), participate in the in vivo production of these ligands in SD
and PV skin. For M. furfur the ability to produce or not these ligands in vitro is a stablebiological trait, as we found out by repeated sub-cultures of our clinical strains and is
exemplary highlighted by the reference strain M. furfur CBS 6094. The latter is a healthyskin isolate and the inability to assimilate L-tryptophan for indole production was noted in 2
independent studies with at least 4 years difference (Mayser et al, 2004; Gaitanis et al,2008).

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The biological effects of the Malassezia-associated ligands found in skin scale extracts were
evaluated in the human hepatoma cell line (CALUX assay) and correlated well with therespective indole quantification. The potency order of the pure indoles and TCDD whenincubated for 6 h was measured as follows: indirubin>FICZ>TCDD>ICZ>malassezin>
tryptanthrin. Regarding skin scale extracts, the lowest EC50s for gene induction wererecorded for those containing the highest concentrations of indirubin with or without FICZ.
For example, in samples SD1 and SD2 where only indirubin was found, the AhR activity
could satisfactorily be explained by the levels of indirubin. In the case of SD2 theconcentration of indirubin corresponding to the EC50 of the extract is 1.2×10−11M which isvery close to the EC50 of pure indirubin (2.6×10−11M). Overall the EC50 for gene induction
from the lesional skin extracts was significantly lower than that measured for the control
skin extracts. The observed notable activation of the AhR from some healthy skin extracts,
without identification of Malassezia indoles in the chemical analysis, could be attributed toenvironmental pollutants or inherent chemical factors with AhR activity found in humanskin.
The clinical significance of the Malassezia produced indoles was further confirmed in theimmortalized epidermal cell HaCaT by examining the AhR inducing ability either of thepure indoles or the M. furfur culture extracts. The tested AhR agonists showed clear ligand,time and dose-dependent effects on CYP1A1, CYP1B1 and ALDH3A1 mRNA expression(Fig. 3 and Fig. S4). FICZ in addition to ICZ and indirubin were found to be the most potent
AhR agonists as suggested from previous reports (Flaveny et al, 2009; Wincent et al, 2009).

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The culture extracts induced statistically significant alterations in AhRR, CYP1A1, CYP1B1
and ALDH3A1, but also in GSTT1 and GSTP1 mRNA expression (Fig. 2, Fig. S3).
Interestingly, AhRR and GSTs mRNA expression was decreased by the extracts while
Aldh3a1 showed variable expression. AhRR down-regulation points towards the existenceof additional bioactive substances while down regulation of Gstp could be attributed totryptanthrin (Yu et al, 2009).

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Our findings do not demonstrate any causal associations, yet this the next step, as theexistence of potent AhR ligands should be complemented in future research with theassessment of the in vivo AhR activation level in the epidermis of Malassezia-associatedskin diseases, as is SD and PV. Indeed, in an experimental model in mice, constitutively
activated AhR resulted in inflammatory skin lesions (Tauchi et al., 2005). In PV and SD,these lipophilic ligands could cross the defective epidermis, reach the granular and spinous
layers, and activate the AhR (Swanson HI, 2004) and cause the respective downstream
effects. The corresponding, AhR mediated local metabolic and immune aberrations havebeen proposed as the pathophysiological background of the suggested Malassezia indoles
and basal cell carcinoma association (Gaitanis et al, 2011). It is noteworthy that ICZ, like
TCDD, has been considered as a tumor promoter (Herrmann et al, 2002). Anotherinteresting issue that rises from our findings is the impact these indoles could have on thelocal induction of P450 enzymes that metabolize antifungal azoles. Although the main azole
metabolizing enzyme CYP3A4 is not directly controlled by AhR (Swanson HI, 2004) there
is increasing evidence that there is a cross-talk between AhR, PXR and CYP3A4 (GerbalChaloin et al, 2006) that could affect the skin metabolism of the commonly used antifungal
azoles. Currently, AhR is also under intense investigation as it can modulate the function of
T-regs and Th17 lymphocytes (Quintana et al, 2008) as well as dendritic cells (Vlachos et at,
2012; Voorhis et al, 2012) in a ligand dependent manner. Morevorer, the Malassezia
produced indirubin and ICZ have been recently shown to inhibit Toll-like receptor induced
maturation of dendritic cells, a finding that coincides with the need of a skin commensal toameliorate local immune reactions in its niche (Vlachos et al, 2012).

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In conclusion, this article describes the synthesis of bioactive indoles by the majority of thecurrently accepted Malassezia species and the preferential production of all the most active
known natural indolic AhR ligands in culture extracts of M. furfur pathogenic strains. Thesefindings coupled by the presence of the AhR ligands in skin scales of Malassezia-associateddiseased skin corroborates indole production as a virulence factor in this yeast. Although therole of Malassezia yeasts in human skin is commonly neglected or underestimated, based on
the observed strong activation of AhR in HaCaT cells by Malassezia extracts -and the pureindolic constituents- we can propose that the presence on the human skin of a
microorganism able to continually synthesize highly potent AhR ligands plays a crucial rolein skin homeostasis and in the development of skin diseases, like SD and PV.

Materials & Methods
Skin sample collection and extraction
Skin scales (50–100 mg) from ten patients (6 SD and 4 PV) were collected and stored at−80°C until extraction. For control samples, scraping of healthy facial skin (N=3) andnormally thicker skin from the elbows, palms and soles (N=3) was performed. All patientsgave a written informed consent. All samples were extracted with EtOAc (10 mL) undersonication (30 min). The resulting solution was filtrated and evaporated under reducedpressure. The residue was dissolved in DMSO to give a stock solution (100 mg/mL skinextract). Subsequently, a series of diluted solutions to 1 μg/mL were prepared for the AhR
test.

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Malassezia growth conditions

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Type strains of 12 Malassezia species and clinical strains of M. furfur were grown in Ltryptophan agar as previously (Gaitanis et al, 2008).
Chemical synthesis
Malassezin, FICZ, ICZ, indirubin and tryptanthrin were synthesized following literature
methods described in supporting information.
Extraction and isolation of Malassezia metabolites from M. furfur cultures
Indirubin, FICZ and tryptanthrin were isolated from the M. furfur Bul22 strain employing
column chromatography with Sephadex LH-20 and preparative HPLC (column: Nucleosil
100-7, RP-18, 7 μm, 250×21 mm) with a gradient system from 100% H2O to 100%
acetonitrile in 180 min, with flow 5 mL/min.
Extraction and HPLC screening
The tested Malassezia strains (Tables 2,3) were grown, extracted and submitted to analytical
HPLC as previously (Gaitanis et al, 2008). The newly identified substances were detectedwith UV as follows: indirubin at 540 nm, ICZ and malassezin at 331 nm, FICZ at 454 nm
and tryptanthrin and pityriacitrin at 390 nm.

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LCMS analysis
The LC/MS/MS analysis was performed using an Agilent 6460 QQQ operating on ESI
mode. A portion of the stock solution (1 μL) was submitted to LCMS (RP Poroshell 120,
150×2.1 mm) using a gradient system from 95% H2O/5% Acetonitrile to 100% acetonitrilein 10 min, with flow 0.3 mL/min (Fig. S1).
HG2L7.5c1 CALUX cell bioassay
HG2L7.5c1 cells were trypsinized and resuspended in 20 mL α-MEM. An aliquot (100 μL)of the suspension was added into 96-well tissue culture plates and incubated for 24h.
Subsequently they were incubated with controls and test compounds for 6h or 24h at 37°C.
Luciferase activity was measured as previously (Baston and Denison 2010).
HaCaT cell analysis

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HaCaT cells were grown in DMEM supplemented with 10% FBS, L-glutamine andstreptomycin/penicillin. The extracts from the clinical M. furfur strains GS19A, Bul22,
Bul412 and WCH106 were normalized at 100 nM ICZ. HaCaT cells were grown to nearly
confluence and treated with the respective extract for 24h. The AhR ligands were also tested
for dose and time response effects on HaCaT cells accordingly. Total RNA was isolated
employing the Nucleospin kit (Macherey-Nagel, Germany).
qRT-PCR
mRNA of the AhR, AhR repressor (AhRR) genes and AhR regulated genes (CYP1A1,
CYP1B1, ALDH3A1, GSTT1 and GSTP1) was measured with a CFX-96-quantitative RTPCR system (BioRad Lab) with Taqman® Universal PCR master mix (4304437, AB, NJ,
USA) using inventoried 20x assay mixes of the respective primer/probe sets as detailed, also
for qRT-PCR conditions in Supporting material. Relative quantification was performed
using the ΔCt method which results in ratios between the target genes and the house keeping
reference gene β-actin (4352935E, AB, NJ, USA).

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Statistical analysis

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For statistical analysis Mann-Whitney U-test and Student’s t-test were employed using
SPSS17 (Chicago, Ill) for analysis. Two-sided p<0.05 values were considered significant.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
We thank Prof. Alyson Mitchell and Prof. Susan Ebeler for providing Eleni Melliou the opportunity to run the
LCMS experiments in the Food safety Laboratory at UC Davis. We also thank Stravroula Kritikou for expert
technical assistance in Malassezia cultures, Guochun He for assistance in CALUX assays, Nikos Lemonakis andEvagelos Gikas for their help in some LCMS experiments. This work was supported in part by the General
Secretariat of Research and Technology of Greece (Program PENED), the National and Kapodistrian University ofAthens (Program Kapodistrias) and the National Institutes of Environmental Health Sciences (ES007685 andES04699).

Abbreviations

NIH-PA Author ManuscriptADatopic dermatitis

AhR

Aryl hydrocarbon receptor

AHRR

AhR repressor

CALUX

Chemical Activated Luciferase gene eXpression

FICZ

formyl-indolo[3,2-b]carbazole

ICZ

indolo[3,2-b]carbazoleMS

Mass Spectroscopy

NMR

Nuclear Magnetic ResonancePVpityriasis versicolor

SD

Seborrheic Dermatitis

TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)

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FIGURE 1.

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AhR induction in Recombinant human hepatoma (HG2L7.5c1) cells after incubation for 6 hmeasured with the CALUX cell bioassay. The results are normalized to the maximal activityof each compound.

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FIGURE 2.

Effects of M. furfur extracts on AhR-dependent genes expression. Alterations in the relative
levels of mRNA expression of the Cyp1a1 (Panel a), Cyp1b1 (Panel b), AhRR (Panel c)genes are shown [mean values ± standard deviations (bars) of 3 independent experiments].
The extracts were normalized to their ICZ content. The asterisk denotes statisticallysignificant differences compared to control which is expressed as 100% (p<0.05; see also
Fig. 3). Bul22, Bul412 and GS19 are strains isolated from seborrheic dermatitis (SD) andWCH106 from Malassezia-folliculitis.

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FIGURE 3.

Comparative induction of CYP1A1 and CYP1B1 (insert) mRNA by FICZ, ICZ, indirubin(IND), malassezin (MAL) and tryptanthrin (TRY) at 24h [mean values ± standard deviations(bars) of 3 independent experiments]. Statistically significant induction of Cyp1a1 is noted
at 100 nM for FICZ, ICZ and indirubin. At 1 μM FICZ is a significantly more potent
Cyp1a1 inducer than ICZ or indirubin. This also applies for Cyp1b1 at 1 μM (insert). The
asterisk denotes statistically significant differences compared to control (p<0.05).

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ND

ND
1.0×10−11
2.1×10−11
ND
ND
ND
ND
ND
ND
ND
ND
ND

ND

2×10−13ND

2.5×10−12

1.1×10−12

ND

ND

ND

ND

ND

ND

ND

SD5

SD6

PV1

PV2

PV3

PV4

Healthy1

Healthy2

Healthy3

Healthy4

Healthy5

Healthy6

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

1.3×10−11

2.6×10−10

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

1.5×10−11

4.1×10−12

ND

ND

7.5×10−11

ND: Not detected; Limits of detection (mol/mg): Malassezin: 4×10−14, ICZ: 8×10−12, FICZ: 2.9×10−13, Indirubin: 6×10−13, Pityriacitrin: 3×10−13

1

ND

ND

ND

4.2×10−12

SD4

ND

5×10−13ND

ND

8.1×10−11
1.5×10−10

Pityriacitrin mol/mg skin extract

Indirubin mol/mg skin extract

1.1×10−11

ND

2×10−13

SD3

ND

ND

FICZ mol/mg skin extract

ND

ND

ND1

ND

ICZ mol/mg skin extract

Malassezin mol/mg skin extract

SD2

SD1

>100

>100

83

100

>100

53

>100

0.09

0.07

4.9

7.9

>100

0.67

0.88

0.08

0.1

EC50 μg/mL

Quantitation (mol/mg of extract) of malassezin, indirubin, indolo[3,2-b]carbazole (ICZ), and 6-formylindolo[3,2-b]carbazole (FICZ) and pityriacitrin by
HPLC/MS/MS in the extracts of skin originating patients with seborrheic dermatitis (SD) or pityriasis versicolor (PV), and healthy subjects (Healthy 1–3:
elbows, palms and soles, Healthy 4–6: facial skin). Values are means of two independent measurements. The EC50 for stimulating AhR-dependent
luciferase activity of each skin extract has been measured by the CALUX cell bioassay.

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Table 1
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SD1
SD
SD
SD
SD
SD
SD
SD
PV2
Folliculitis
HS3
HS
HS
HS
HS
HS
HS

M. furfur Bul19

M. furfur Bul22

M. furfur Bul23

M. furfur Bul412

M. furfur CBS9585

M. furfur CBS9596

M. furfur GS19A

M. furfur WCH114

M. furfur WCH100

M. furfur WCH106

M. furfur GS1B

M. furfur GS2A

M. furfur GS2B

M. furfur GS4A

M. furfur GS46A

M. furfur GS9A

M. furfur GA9B

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ND: Not detected4

HS: healthy subject;

PV: Pityriasis versicolor;

32

SD: Seborrheic dermatitis;

1

Skin of origin of the sample

Strain0.43±0.04

0.56±0.04

0.45±0.02

0.39±0.04

0.39±0.04

0.40±0.03

0.38±0.03

5.99±0.51

3.17±0.37

4.21±0.40

2.16±0.15

4.12±0.25

2.02±0.20

2.02±0.18

3.52±0.29

3.33±0.38

4.08±0.35

Malassezin (μg/mg)

0.008±0.002

0.026±0.002

0.010±0.001

0.009±0.002

0.009±0.002

0.006±0.001

0.006±0.001

1.48±0.14

1.58±0.14

1.37±0.15

0.022±0.01

2.66±0.21

1.40±0.13

3.36±0.34

0.25±0.02

3.90±0.33

0.084±0.010

Indirubin (μg/mg)

ND

ND

ND

ND

ND

ND

ND4

3.27±0.29

0.82±0.05

1.47±0.11

0.38±0.02

1.60±0.12

0.40±0.02

0.95±0.07

0.36±0.02

2.50±0.11

0.26±0.01

ICZ (μg/mg)

0.088±0.07

0.37±0.02

0.19±0.01

0.16±0.01

0.094±0.007

0.031±0.005

0.029±0.005

4.50±0.38

1.65±0.11

0.66±0.05

0.05±0.01

0.58±0.03

0.24±0.02

0.53±0.03

0.38±0.03

1.52±0.09

0.59±0.03

Tryptanthrin (μg/mg)

ND

ND

ND

ND

ND

ND

ND

0.57±0.05

0.67±0.04

0.58±0.03

ND

0.68±0.06

0.73±0.06

0.72±0.05

0.45±0.04

1.14±0.10

1.33±0.15

FICZ (μg/mg)

0.68±0.05

0.35±0.05

0.77±0.06

1.18±0.09

0.55±0.03

0.57±0.03

0.41±0.04

8.27±0.45

3.07±0.28

0.75±0.35

2.49±0.25

2.31±0.23

4.93±0.35

2.76±0.27

3.63±0.28

3.62±0.30

4.82±0.33

Pityriacitrin (μg/mg)

Quantitation (μg/mg of extract) of malassezin, indirubin, indolo[3,2-b]carbazole (ICZ), tryptanthrin and 6-formylindolo[3,2-b]carbazole (FICZ) by
HPLC/UV in the extracts of clinical M. furfur strains originating from seborrheic dermatitis, pityriasis versicolor, folliculitis and healthy subjects. Values
are means and standard deviations of three independent measurements.

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Table 2
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Traces2
TracesTracesND
Traces
0.30±0.02
0.34±0.03
0.29±0.02
ND

M. obtusa CBS7876

M. globosa CBS7966

M. slooffiae CBS7971

M. restricta CBS7991

M. dermatis CBS9170

M. japonica CBS9432

M. nana CBS9557

M. yamatoensis CBS9726

M. caprae CBS10434

Trace: detectable but not quantifiable

2

ND: Not detected;

1

ND1

M. sympodialis CBS7222

3.52±0.25

M. furfur CBS6001
0.28±0.02

2.02±0.18

M. furfur CBS1878

M. pachydermatis CBS6534

Malassezin (μg/mg)

Malassezia species

ND

0.002±0.001

ND

0.005±0.001

ND

ND

ND

0.006±0.001

0.006±0.001

ND

0.002

ND

0.062±0.005

Indirubin (μg/mg)

ND

Traces

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

0.42±0.03

ICZ (μg/mg)

ND

TracesND

0.017±0.02

ND

ND

ND

0.074±0.04

TracesND

TracesND

0.49±0.04

Tryptanthrin (μg/mg)

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

0.84±0.02

FICZ (μg/mg)

ND

0.24±0.04

ND

0.33±0.05

ND

ND

ND

ND

ND

ND

ND

ND

0.75±0.05

Pityriacitrin (μg/mg)

Quantitation (μg/mg of extract) of malassezin, indirubin, indolo[3,2-b]carbazole (ICZ), tryptanthrin and 6-formylindolo[3,2-b]carbazole (FICZ) by
HPLC/UV in the extracts of 13 Type and reference Malassezia species strains. Values are means and standard deviations of three independentmeasurements.

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Table 3
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