A Small Aromatic Compound Has Antifungal Properties and Potential Anti-Inﬂammatory Effects Against Intestinal Inﬂammation

International Journal of Molecular Sciences

Article A Small Aromatic Compound Has Antifungal Properties and Potential Anti-Inﬂammatory Effects against Intestinal Inﬂammation

Clovis Bortolus 1,2,3, Muriel Billamboz 2,4 , Rogatien Charlet 1,2,3, Karine Lecointe 1,2,3 , Boualem Sendid 1,2,3, Alina Ghinet 2,4,5 and Samir Jawhara 1,2,3,*

1 Institut National de la Santé et de la Recherche Médicale, U995/Team2, F-59000 Lille, France; [email protected] (C.B.); [email protected] (R.C.); [email protected] (K.L.); [email protected] (B.S.) 2 Lille Inﬂammation Research International Center, University Lille, U995-LIRIC, F-59000 Lille, France; [email protected] (M.B.); [email protected] (A.G.) 3 Service de Parasitologie Mycologie, Pôle de Biologie Pathologie Génétique, CHU Lille, F-59000 Lille, France 4 Laboratoire de Chimie Durable et Santé, Ecole des Hautes Etudes d’Ingénieur (HEI), Yncréa Hauts-de-France, 13 Rue de Toul, F-59046 Lille, France 5 Faculty of Chemistry, Al. I. Cuza’ University of Iasi, B-dul Carol 1 nr. 11, 700506 Iasi, Romania * Correspondence: [email protected]; Tel.: +33-(0)3-20-62-35-46; Fax: +33-(0)3-20-62-34-16

Received: 7 December 2018; Accepted: 4 January 2019; Published: 14 January 2019

Abstract: Resistance of the opportunistic pathogen Candida albicans to antifungal drugs has increased significantly in recent years. After screening 55 potential antifungal compounds from a chemical library, 2,3-dihydroxy-4-methoxybenzaldehyde (DHMB) was identified as having potential antifungal activity. The properties of DHMB were then assessed in vitro and in vivo against C. albicans overgrowth and intestinal inflammation. Substitution on the aromatic ring of DHMB led to a strong decrease in its biological activity against C. albicans. The MIC of DHMB was highly effective at eliminating C. albicans when compared to that of caspofungin or fluconazole. Additionally, DHMB was also effective against clinically isolated fluconazole- or caspofungin-resistant C. albicans strains. DHMB was administered to animals at high doses. This compound was not cytotoxic and was well-tolerated. In experimental dextran sodium sulphate (DSS)-induced colitis in mice, DHMB reduced the clinical and histological score of inflammation and promoted the elimination of C. albicans from the gut. This finding was supported by a decrease in aerobic bacteria while anaerobic bacteria populations were re-established in mice treated with DHMB. DHMB is a small organic molecule with antifungal properties and anti-inflammatory activity by exerting protective effects on intestinal epithelial cells.

Keywords: Candida albicans; 2,3-dihydroxy-4-methoxyBenzaldehyde; melanin; colitis; anaerobic bacteria; aerobic bacteria

1. Introduction Inﬂammatory bowel diseases (IBDs), comprising Crohn’s disease (CD) and ulcerative colitis, have a multifactorial aetiology with complex interactions between genetic susceptibility, the immune system, environment and the microbiota [1]. The biodiversity of the gut microbiota is frequently decreased in IBD patients, in particular a reduction in Firmicutes and an increase in Proteobacteria are often observed [2]. Escherichia coli is part of the Proteobacteria phylum and is increased greatly while Lactobacillus species belonging to the Firmicutes phylum are reduced in IBD patients [3,4]. Other

Int. J. Mol. Sci. 2019, 20, 321; doi:10.3390/ijms20020321 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 321 2 of 14 observations supporting a role for the gut microbiota in IBD include an abundance of fungi in the inflamed mucosa and faeces [5,6]. Colonisation with Candida albicans, an opportunistic human fungal pathogen, is consistently increased in CD patients [7]. In addition, abundance of this fungus is correlated with serological markers of CD, namely anti-Saccharomyces cerevisiae antibodies (ASCA) [5,8]. Recently, in an experimental murine model of dextran sodium sulphate (DSS)-induced colitis, the population of aerobic bacteria, in particular E. coli and Enterococcus faecalis, increased, whereas the population of anaerobic bacteria such as Lactobacillus johnsonii and Bifidobacterium species decreased. These microbiota modifications were associated with overgrowth of fungi suggesting that dysbiosis could play a crucial role in IBD [9]. Different anti-inflammatory compounds including 5-aminosalicylic acid (5-ASA) or anti-TNFα antibodies have been used to treat IBD patients, but no compounds have a dual effect of antifungal and anti-inflammatory activity [10]. Some of the anti-inflammatory drugs are not effective at treating active CD or preventing disease relapse; they only treat the symptoms and are sometimes only effective for a few years [11]. In parallel, an increase in antifungal resistance is considered a major problem to clinicians and leads to high morbidity and mortality rates in patients with invasive mycoses [12]. The increase of new fungal species that are resistant to different classes of available antifungal drugs constitutes an urgent need for developing new antifungal compounds [13]. In the current study, we assessed the in vitro and in vivo antifungal properties of a novel compound, 2,3-dihydroxy-4-methoxybenzaldehyde (DHMB), against C. albicans colonisation and intestinal inflammation.

2. Results

2.1. Structure and Characterisation of DHMB A series of 55 compounds from the private chemical library of the laboratory was screened for antifungal and antibacterial activities, revealing that DHMB was a highly effective molecule against C. albicans. The DHMB MIC, at which C. albicans growth was fully inhibited, was 8 µg/mL. From a chemical point of view, DHMB is a small organic molecule (M.W. = 168.15 g/mol) bearing a highly reactive aromatic aldehyde moiety and two free phenol groups (Figure1). We then evaluated some derivatives of DHMB for their antifungal activity; the structure and activities of these derivatives are summarised in Table1. The biological activity of these compounds against C. albicans was evaluated at 4 × MIC (32 µg/mL). The aldehyde group in position 1 was modified with little or no change in positions 2–5 on the aromatic moiety (Table1, entries 1–6). Deletion of the aldehyde substituent led to a total loss of activity (Compound 1, Table1, entry 2). Compounds 2 and 3, in which the carbonyl group in position 1 is still present but included in a phenylethanone moiety, exhibited weak inhibition at 32 µg/mL (13% and 27%, respectively). Moreover, as exemplified by compounds 4 and 5, modification of the phenolic hydroxyl groups in positions 2 and 3 led to a loss of activity (Table1, entries 5–6). Rigidification of the system and introduction of a lactone ring (compound 6) significantly decreased the biological activity (Table1, entry 7). From these data, the aldehyde group in position 1 seems to be essential for activity against C. albicans. The importance of the catechol and methoxy groups on the aromatic ring was also studied. Unexpectedly, replacement of the 4-methoxy substituent by a hydroxyl one led to a complete loss of activity (Table1, entry 8). This shows that this para-methoxy group plays an essential role in the biological activity, possibly due to its hydrophobicity compared with the hydroxyl moiety. Modification of position 6 was then undertaken whilst retaining the 2,3-dihydroxy-4-methoxy- sequence on the aromatic ring. A carboxylic acid was introduced in that position (3,4-dihydroxy-5-methoxybenzoic acid, compound 8), but no activity against C. albicans was detected, and modifications of the hydrophilicity/hydrophobicity of benzoic acid derivatives did not result in a gain in activity (compounds 9 and 10, Table1). Passing from benzoic acid to benzylic acid did not improve the Int. J. Mol. Sci. 2019, 20, 321 3 of 14 activity (compare compounds 9 and 11, Table1, entries 10 and 12). A similar conclusion can be drawn on replacement of the carboxylic acid by an ethoxy substituent at position 6 (compound 12). In the same way, replacingInt. J. Mol. Sci. the2019, 20 carboxylic, 321 acid by an aldehyde, an acetate or a hydrogen group3 of 15 did not improveInt.Int. the J. J. Mol. Mol. activity Sci. Sci. 2019 2019, , (comparison20 20, ,321 321 of compound 10 with compounds 13–15). 33 of of 15 15 Int. J. Mol. Sci. 2019, 20, 321 3 of 15

Figure 1. Chemical structure of DHMB and related investigated compounds. Figure 1. ChemicalFigure 1. Chemical structure structure of DHMB of DHMB and and related related investigated compounds. compounds. Figure 1. Chemical structure of DHMB and related investigated compounds. Figure 1. ChemicalTable 1. Chemical structure modifications of DHMB ofand DHMB related and investigated their impact on compounds. activity. Table 1. ChemicalTable 1. Chemical modiﬁcations modifications ofDHMB of DHMB and and theirtheir impact impact on onactivity. activity. Table 1. Chemical modifications of DHMB and their impact on activity.% Inhibition at 32 Entry CompoundTable 1. Chemical R1 modificationsR2 of DHMBR3 and theirR4 impactR5 on Ractivity.6 µg/mL% aInhibition at 32 Entry Compound R1 R2 R3 R4 R5 R6 % Inhibition Entry Compound1 DHMB R1 CHO R2 OH R3 OH OCHR43 H HR 5 % RInhibition 1006 µg/mL at 32 a a Entry Compound R1 R2 R3 R4 R5 R6 % Inhibition atat 32 32 µg/mL Entry Compound2 1 R1 H R2 OH R3 OH ROCH4 3 RH5 HR 6 3 a 1 DHMB CHO OH OH OCH3 H H µg/mLµg/mL a 100 11 DHMB CHO CHO OH OH OH OH OCH OCH3 3 H 3 H H H 100 100 1 2 DHMB 1 CHO H OH OH OH OHOCH 3 OCHH H H H 100 3 3 2 OH OCH3 OCH3 H H 13 22 1 H H OH OH OH OH OCH OCH3 3 H H H H 3 3 2 1 H OH OH OCH3 H H 3

3 3 3 4 2 3 OHOH OHOCH OCH3 OCHH H H H 27 13 33 2 OHOH OCHOCH3 OCHOCH3 H H H H 13 13 3 52 4 OH OH OCHOCOCH3 2Cl OCHOCH3 3 3HH H H 6 13 4 3 OH OH OCH3 H H 27 6 5 OCOCH2Cl OCOCH2Cl OCH3 H H 4 4 5 3 4 OH OH OHOCOCH OCH2Cl 3 OCHH 3 H H H 27 6 44 3 3 OHOH OHOH OCH OCH3 3H H H 27 27 5 4 OH OCOCH2Cl OCH3 H H 6 55 4 4 OH OH OCOCHOCOCH2ClCl OCHOCH3 3H H H 6 6 6 5 OCOCH2Cl OCOCH2Cl OCH3 H H 4 6 5 OCOCH Cl OCOCH Cl OCH H H 4 6 7 5 6 OCOCH2 2Cl OCOCH22Cl OCH3 3 H H H 2 4 6 5 OCOCH2Cl OCOCH2Cl OCH3 H H 4

77 8 6 6 7 CHO OH OH OH H H H H 4 2 2 9 8 H OH OH OCH3 H COOH 2 77 66 HH 22 10 9 H OCH3 OCH3 OCH3 H COOH 2

11 10 H OCH3 OH OCH3 H COOH 0 8 7 CHO OH OH OH H H 4 8 7 CHO OH OH OHCH H2- H 4 8 9 12 7 8 11 CHO H OH OCH OH3 OHOCH 3OH OCHOH 3 OCHH 3 H H COOH 1 4 2 98 7 8 CHO H OH OH OH OH OH OCH 3H COOHH COOH 4 2 9 10 8 9 H H OH OCH3 OH OCH3OCH 3 OCHH 3 H COOH COOH 2 2 109 138 912 H H H OCH OH3 OCH3 OCH OHOCH 3 3 OCHOCHOCH3 3 3HH CH COOH2H-OH COOH2 2 2 10 11 14 9 1013 H H OCH OCH3 OCH3 3 OCHOH3 OH OCHOCH33 OCHHH 3 H CHO COOH COOH 1 2 0 1110 9 10 H H OCH OCH3 3 OCHOH3 OCH OCH3 3H COOHH COOH 2 0 11 1510 14 H H OCH OCH3 3 OHOH OCHOCH33 HH COCH COOH3 2 4 0 3 3 CH - 1211 12 10 11 11 H H H OCH OCH3 OCH3 OCHOH 3 OCHOCH3 OCH OCH3H 3 COOHH H CH2-COOH 0 1 1 16 15 H OCH3 OH OCH3 H CH H 2- COOH 0 1312 11 12 H H OCH OCH3 OCHOCH3 OCHOCH3 H CHH2- CH -OH1 2 a 3 3 3 3 3 2 12 13 11 12 H H Screening OCH carried OCH out3 OCH on C. albicansOCHOCH3 ATCC OCH H90028. 3 HCOOH CH 2-OH 1 2 14 13 H OCH3 OH OCH3 COOHH CHO 1 13 14 12 13 H H OCH3 OCH3 OCH3 OH OCH3 OCHH 3 CHH 2-OH CHO 2 1 1513 12 14 H H OCH OCH3 3 OCHOH3 OCH OCH3 3H CHH2-OH COCH32 4 14 15 13From 14 these data, H the aldehyde H OCH group3 OCH in 3position OH 1OH seems OCH to3 OCH beH essential 3 H CHO for COCH activity3 against 1 C.4 1614 13 15 H H OCH OCH3 3 OHOH OCH OCH3 3H CHOH H 1 0 15 14 H OCH3 3 OH OCH3 H 3 COCH3 4 15 16albicans 14 . The15 importance H of H the catechol OCH3 and OCH methoxy OH grOHoupsOCH on3 theOCHH aromatic COCHH ring3 Hwas also studied.4 0 a 16 15 H a OCH3 OHC. albicansOCH3 H H 0 16 Unexpectedly,15 replacement H Screening of Screening the OCH4-methoxy carried3 carried out substituen out onOH on C.t byalbicansOCH a hydroxyl3ATCC ATCCH 90028.one 90028. Hled to a complete 0 loss of a activity (Table 1, entrya Screening Screening8). This showscarried carried that out out thison on C. C.para albicans albicans-methoxy ATCC ATCC group 90028. 90028. plays an essential role in the biologicalFrom these activity, data, possibly the aldehyde due to groupits hydrophobicity in position 1compared seems to withbe essential the hydroxyl for activity moiety. against C. FromFromalbicansModification these these. The data, data, importance of the theposition aldehyde aldehyde of 6 thewas group groupcatechol then in undertakenin position andposition methoxy whilst1 1 seems seems gr retainingoups to to be be on essential essentialthe the 2,3-dihydroxy-4-methoxy- aromatic for for activity activity ring was against against also studied.C. C. 2.2. In Vitro Antifungalsequence on Activity the aromatic of DHMBring. A carboxylic against acid C. was albicans introduced in that position (3,4-dihydroxy-5- albicansalbicansUnexpectedly,. .The The importance importance replacement of of the the catechol catechol of the 4-methoxyand and methoxy methoxy substituen gr groupsoups ont on by the thea hydroxyl aromatic aromatic onering ring led was was to also aalso complete studied. studied. loss of Unexpectedly,methoxybenzoic replacement acid, of compound the 4-methoxy 8), but substituen no activityt by against a hydroxyl C. albicans one led was to adetected, complete and loss of TheUnexpectedly, antifungalactivitymodifications (Table replacement activity 1,of theentry of hydrophilicity/hydrophobicity of DHMB 8).the This 4-methoxy againstshows substituenthatC. this albicans of para tbenzoic by-methoxy awas hydroxyl acid compared derivatives group one ledplays did withto not aan completeresult essential that in ofa gain loss caspofunginrole of in the and activity (Table 1, entry 8). This shows that this para-methoxy group plays an essential role in the fluconazole.activitybiological The (Tablein activity viability 1, activity, entry(compounds of8). possiblyC. This 9 albicans and shows 10, due Table thatcells to 1). thisits Passing was hydrophobicitypara tracked fr-methoxyom benzoic in group acid realcompared to playsbenzy time lican usingwith acid essential didthe a not fungalhydroxyl improverole in bioluminescent the moiety. biologicalbiologicalModificationthe activity, activity,activity (compareof possiblypossibly position compounds due due6 was toto its9thenits and hydrophobicityhydrophobicity 11,undertaken Table 1, entries whilst comparedcompared 10 andretaining 12). with withA similarthe thethe 2,3-dihydroxy-4-methoxy- conclusion hydroxylhydroxyl can moiety.moiety. be µ × strain challengedModificationModificationsequencedrawn with ofof onon position positionreplacement eitherthe aromatic 6 DHMB,6 was wasof thering. thenthen carboxylic caspofunginA undertaken undertakencarboxylic acid by acid anwhilstwhilst or ethoxy was fluconazole retaining retaining introducedsubstituent thethe at atin position2,3-dihydroxy-4-methoxy- 2,3-dihydroxy-4-methoxy- theirthat position 6 MICs (compound (3,4-dihydroxy-5- (8 12).g/mL), 2 MICs (16 µg/mL)sequencesequence ormethoxybenzoic 4 on on× the MICsthe aromatic aromatic (32 acid,µ ring. ring.g/mL) compound A A carboxylic carboxylic utilising 8), acid acidbut different waswasno introduced introducedactivity exposure against in in that that times: C.position position albicans 0, 15, (3,4-dihydroxy-5- (3,4-dihydroxy-5- 30was and detected, 60 min and (Figure 2). methoxybenzoic acid, compound 8), but no activity against C. albicans was detected, and DHMBmethoxybenzoic led tomodifications a significant acid, of the reductioncompound hydrophilicity/hydrophobicity in8), bioluminescencebut no activity of against benzoic as well C.acid asalbicans derivatives in the was number did detected, not result of and fungal in a gain colonies modifications of the hydrophilicity/hydrophobicity of benzoic acid derivatives did not result in a gain indicatingmodifications rapidin activity fungicidal of (compoundsthe hydrophilicity/hydrophobicity activity 9 and after 10, Table incubation 1). Passing of benzoic with fromC. benzoic acid albicans derivatives acid. to This benzy did fungicidal notlic acid result did in activitynota gain improve of DHMB inin activity activitythe activity (compounds (compounds (compare 9 9 and and compounds 10, 10, Table Table 1). 1). 9 Passing andPassing 11, fr Tablefromom benzoic benzoic1, entries acid acid 10 to toand benzy benzy 12).liclic A acid acidsimilar did did notconclusion not improve improve can be was similarthethe activity activity todrawn that (compare (compareon of replacement caspofungin compounds compounds of the 9 while 9 carboxylicand and 11, 11, fluconazole Table Table acid 1, 1,by entries entries an ethoxy exhibited 10 10 and and substituent 12). 12).a A A fungistatic similar similar at position conclusion conclusion effect.6 (compound can can The be be antifungal12). activitydrawn ofdrawn DHMB on on replacement replacement was assessed of of the the oncarboxylic carboxylic drug-resistant acid acid by by an anC. ethoxy ethoxy albicans substituent substituentclinical at at isolatesposition position 6 (Figure6 (compound (compound2E,F). 12). 12). The viability of drug-resistant C. albicans clinical isolates was significantly reduced after treatment with DHMB at 10 × MIC in terms of viable colony counts using fungal culture media (Figure3). To assess whether DHMB has an impact on modulation of the glycan cell wall, flow cytometry analysis and confocal microscopic observations were performed. Confocal microscopy revealed that, in contrast to fluconazole and DHMB, caspofungin challenge for 1 h produced a damaged polysaccharide cell Int. J. Mol. Sci. 2019, 20, 321 4 of 14 wall; in particular, the β-glucan and chitin layers were mainly affected by this antifungal treatment (Figure3A). Similarly, flow cytometry analysis showed that treatment with either fluconazole or DHMB did not affect the fungal cell wall when compared to untreated C. albicans while C. albicans challenged with caspofungin exhibited a significant increase in chitin levels when compared to unchallenged C. albicans. No changes in mannan levels were observed in C. albicans challenged with either fluconazole, caspofungin or DHMB (Figure3B,C). To determine whether DHMB has an impact on the melanisation, which is involved in the virulence of C. albicans, in particular resistance to antifungal agents and protection against oxidative stresses, microscopic observations were realised. A remarkable reduction in the melanin production was observed in C. albicans treated with DHMB when compared to that treatedInt. J. Mol. with Sci. 2019 caspofungin, 20, 321 (Figure3D). 5 of 15

FigureFigure 2. 2. ImpactImpact of of DHMB DHMB on onC. albicansC. albicans viabilityviability.. (A–C) (BioluminescentA–C) Bioluminescent C. albicansC. strain albicans wasstrain treated was treatedwith either with DHMB, either DHMB, flucanoazole flucanoazole or caspofungin or caspofungin at their MICs, at their 2 × MICs MICs, or 2 4× ×MICs MICs orand 4 monitored× MICs and monitoredat 0, 15, 30 at and 0, 15, 60 30 min. and Control 60 min. Controlcorresponds corresponds to C. albicans to C. albicansalone withoutalone without antifungal antifungal treatment; treatment; (D) (DVisualisation) Visualisation of DHMB of DHMB effect effect on onbioluminescent bioluminescent C. albicansC. albicans strainstrain in real in realtime time.. Bioluminescent Bioluminescent C. C.albicans albicans strainstrain was was treated treated with with either either DHMB, DHMB, fluconazo fluconazolele or or caspofungin caspofungin at attheir their MICs, MICs, 2 × 2 MICs× MICs or or 44× ×MICs MICs andand monitored at 0, 15, 15, 30 30 and and 60 60 mi min.n. Line Line 1, 1, PBS PBS without without yeasts; yeasts; Line Line 2, 2,C. C.albicans albicans alonealone;; LinesLines 3–5, 3–5,C. C. albicans albicanstreated treated with withcaspofungin caspofungin atat 11 ×× MIC,MIC, 2 2 ×× MICMIC and and 4 4× ×MIC,MIC, respectively; respectively; Lines Lines 6–8,6–8,C. C. albicans albicanschallenged challenged with fluconazole fluconazole at at 11 ×× MIC,MIC, 2 × 2 MIC× MIC and and 4 × 4MIC,× MIC, respectively; respectively; Lines Lines9– 9–11,11, C.C. albicans albicans + +DHMB DHMB at at1 x 1 xMIC, MIC, 2 × 2 MIC× MIC and and 4 × 4MIC,× MIC, respectively. respectively. * p < *0.0001;p < 0.0001; (E,F) (EffectE,F) Effect of ofDHMB DHMB on on drug-resistant drug-resistant C.C. albicans albicans clinicalclinical isolates isolates.. Control Control represents represents C. C.albicans albicans clinicalclinical isolate. isolate. CaspoCaspo (caspofungin), (caspofungin), FlucoFluco (fluconazole)(fluconazole) or DH DHMBMB (2,3-dihydroxy-4-methoxybenzaldehyde) (2,3-dihydroxy-4-methoxybenzaldehyde) were were addedadded to toC. C. albicans albicansstrains strains atat 1010 × MIC.MIC.

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FigureFigure 3. EffectEffect of of DHMB DHMB on modulationon modulation of chitin of andchitinα-mannan and α-mannan expression. expression. (A) Confocal (A) microscopic Confocal microscopicobservation observation after antifungal after challenge. antifungalC. challenge. albicans was C. treated albicans with was PBS treated (a) caspofungin with PBS (a ()b caspofungin), fluconazole µ ((bc),) orfluconazole DHMB (d ()c) at or a DHMB final concentration (d) at a final ofconcentration 100 M for of 1 h100 and µM the for fungal 1 h and cell the wall fungal was cell analysed wall wasby confocalanalysed microscopy by confocal and microscopy flow cytometry; and flow (B cytometry;) Percentage (B of) Percentage viable C. albicans of viablecells C. labelledalbicans cells with α labelledconcanavalin with A.concanavalin-Mannans wereA. α-Mannans labelled with were concanavalin labelled with A-rhodamine; concanavalin (C) PercentageA-rhodamine; of viable (C) C. albicans cells labelled with WGA-IFTC. Chitin was labelled with wheat germ agglutinin-fluorescein Percentage of viable C. albicans cells labelled with WGA-IFTC. Chitin was labelled with wheat germ isothiocyanate (WGA-IFTC); (D) Microscopic observation of the melanin pigments in C. albicans after agglutinin-fluorescein isothiocyanate (WGA-IFTC); (D) Microscopic observation of the melanin DHMB treatment. (a) C. albicans cells grown without DOPA; (b) C. albicans grown with 1 mM DOPA; pigments in C. albicans after DHMB treatment. (a) C. albicans cells grown without DOPA; (b) C. albicans (c) C. albicans cells grown with DOPA and treated with caspofungin; (d), C. albicans cells were incubated grown with 1 mM DOPA; (c) C. albicans cells grown with DOPA and treated with caspofungin; (d), with DOPA and challenged with DHMB. Bars 10 nm. C. albicans cells were incubated with DOPA and challenged with DHMB. Bars 10 nm. 2.3. Effect of DHMB on C. albicans Colonisation and Intestinal Inflammation in DSS-Induced Colitis Model 2.3. .Effect of DHMB on C. albicans Colonisation and Intestinal Inflammation in DSS-Induced Colitis Model To assess whether DHMB can be tolerated in mice, a high dose of DHMB (100 mg/kg) was injectedTo assess intraperitoneally whether DHMB in mice. can No be mortalitytolerated wasin mice, observed a high following dose of DHMB DHMB challenge (100 mg/kg) (data was not injectedshown). intraperitoneally Body weight remained in mice. stable No mortality and no clinical was observed signs were following observed DHMB over twochallenge weeks. (data not shown).The Body efficacy weight of DHMB remained at eliminatingstable and noC. cl albicansinical signswas were assessed observed in a DSS-induced over two weeks. colitis model. AfterTheC. albicansefficacychallenge, of DHMB DHMB at eliminating was administered C. albicans for was five assessed days (10 in mg/kg) a DSS-induced to assess thecolitis therapeutic model. Afterproperties C. albicans of this challenge, compound. DHMB As was the administered DHMB compound for five has days fungicidal (10 mg/kg) activity to assess against the therapeuticC. albicans , propertiesits effectiveness of this wascompound. compared As the to that DHMB of caspofungin compound has in termsfungicidal of C. activity albicans againstelimination C. albicans from, theits effectivenessgut. No sign was of inflammation compared to wasthat observedof caspofungin in mice in receivingterms of C. water albicans (CTL), eliminationC. albicans fromonly the (Ca) gut. or NoDHMB sign of (Figure inflammation4). Following was observed DSS treatment in mice alone receiving (D) or waterC. albicans (CTL), C.challenge albicans only with (Ca) DSS or treatment DHMB (Figure(CaD), mice4). Following showed DSS a gradual treatment reduction alone in(D) body or C. mass albicans over challenge two weeks, with whereas DSS treatment CaD mice (CaD), receiving mice showedDHMB showeda gradual less reduction severe weight in body loss mass than thoseover two untreated weeks, or whereas treatedwith CaD caspofungin mice receiving (CaDCaspo) DHMB showed(Figure 4lessA). severe The clinical weight score loss forthan inflammation, those untreated including or treated diarrhoea with caspofungin and rectal (CaDCaspo) bleeding, decreased (Figure 4A).significantly The clinical in CaD score mice for treated inflammation, with DHMB including while caspofungindiarrhoea and treatment rectal bleeding, did not significantly decreased significantlydecrease these in clinicalCaD mice symptoms treated with (Figure DHMB4B). Histological while caspofungin analysis treatment of colon samplesdid not showedsignificantly that decreaseDHMB treatment these clinical significantly symptoms reduced (Figure disease 4B). Hist severityological in CaD analysis mice whenof colon compared samples to showed those treated that DHMBwith caspofungin treatment significantly (Figure4C). reduced disease severity in CaD mice when compared to those treated with caspofungin (Figure 4C).

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FigureFigure 4. 4. DeterminationDetermination of of inflammatory inflammatory parameters parameters after after DHMB DHMB treatment treatment in in a a DSS-induced DSS-induced colitis colitis model.model. (A (A) )Body Body weight weight loss loss during during colitis colitis development. development. CTL CTL refers refers to to the the control control group group receiving receiving water.water. Ca Ca corresponds corresponds to to mice mice receiving receiving C.C. albicans albicans alone.alone. DHMB DHMB refers refers to to mice mice treated treated with with DHMB DHMB only.only. D correspondscorresponds to to mice mice receiving receiving DSS DSS only. only DCa. correspondsDCa corresponds to mice to receiving mice receiving DSS and challengedDSS and challengedwith C. albicans with. DCaCaspoC. albicans. andDCaCaspo DCaDHMB and refersDCaDHMB to mice refers receiving to mice DSS receiving and challenged DSS and with challengedC. albicans withand C. treated albicans with and either treated caspofungin with either or caspofungin DHMB (* p or< 0.05);DHMB (B ()* Clinicalp < 0.05); score (B) Clinical for inflammation; score for inflammation;(C) Histological (C) score Histological for inflammation. score for inflammation. Data are the Data mean are± theSD mean of 10 ± mice SD of per 10 groupmice per from group two fromindependent two independent experiments. experiments.

InIn contrast contrast to to CaD CaD or or CaDCaspo, CaDCaspo, colon colon sections sections from from the the CaDDHMB CaDDHMB group group showed showed low low levels levels ofof leukocyte leukocyte infiltrates, infiltrates, oedema oedema and and cryptic cryptic abscesses, abscesses, and and the the surface surface epithelia epithelia structures structures remained remained intactintact (Figure (Figure 5).5). The number of C. albicans colony-forming units (CFUs) and microbiota changes were assessed in freshly collected stool samples from each tagged mouse. A high number of C. albicans CFUs was recovered from stools from all groups on Day 1 (Figure6). In the absence of DSS, C. albicans was dramatically decreased in mice. DSS treatment promoted a significant increase in C. albicans CFUs starting on Day 12 while DHMB administration favoured C. albicans elimination in DSS treated mice. The number of C. albicans CFUs was assessed in the stomach, caecum and colon (Figure6). Treatment with either caspofungin or DHMB promoted C. albicans elimination from the mouse gut. For the gut microbiota, high numbers of E. coli and E. faecalis populations were observed following DSS treatment. Overgrowth of these two populations occurred regardless of C. albicans colonisation (Figure7). DHMB treatment significantly maintained low levels of E. coli and E. faecalis populations while caspofungin treatment failed to reduce them. In terms of anaerobic bacteria, the number of L. johnsonii colonies was significantly reduced in both DSS and C. albicans mice, while DHMB treatment significantly restored L. johnsonii population in colitic mice challenged with C. albicans. In contrast, L. reuteri population showed unpredictable changes (Figure7). The expression levels of pro-inflammatory cytokine IL-1 β and anti-inflammatory cytokine IL-10 were assessed in the colons (Figure8A,B). Expression of IL-1 β was significantly lower in the colons of CaD mice treated with DHMB than in CaD or DSS mice

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(Figure8A,B). In contrast, expression of IL-10 was signiﬁcantly higher in colons of CaD mice challenged with DHMB when compared to that of CaD or DSS mice. Additionally, the expression levels of TLR-4 and TLR-8 were determined. TLR-4 and TLR-8 expression increased signiﬁcantly in response to both C. albicans overgrowth and colitis while treatment with DHMB decreased the expression of these receptorsInt. J. Mol. inSci. the 2019 colons, 20, 321 (Figure 8A,B). 8 of 15

Figure 5. Analysis of histological colon sections from DSS-induced colitis: (a–c) colon sections from Figure 5. Analysis of histological colon sections from DSS-induced colitis: (a–c) colon sections from mice receiving water (control), C. albicans alone and DHMB, respectively; (d) colon sections from mice receiving water (control), C. albicans alone and DHMB, respectively; (d) colon sections from mice mice receiving DSS; (e) colon sections from mice receiving C. albicans and DSS; and (f) colon sections receiving DSS; (e) colon sections from mice receiving C. albicans and DSS; and (f) colon sections from from CaD mice treated with DHMB; (g–i) colon sections from either DSS mice or CaD show tissue CaD mice treated with DHMB; (g–i) colon sections from either DSS mice or CaD show tissue destruction, an important inﬂammatory cell inﬁltrate and oedema in the mucosa and submucosa of destruction, an important inflammatory cell infiltrate and oedema in the mucosa and submucosa of colon wall structures (*). Scale bars represent 50 µm (a–f) and 10 µm (g–i). colon wall structures (*). Scale bars represent 50 µm (a–f) and 10 µm (g–i).

The number of C. albicans colony-forming units (CFUs) and microbiota changes were assessed in freshly collected stool samples from each tagged mouse. A high number of C. albicans CFUs was recovered from stools from all groups on Day 1 (Figure 6). In the absence of DSS, C. albicans was dramatically decreased in mice. DSS treatment promoted a significant increase in C. albicans CFUs starting on Day 12 while DHMB administration favoured C. albicans elimination in DSS treated mice. The number of C. albicans CFUs was assessed in the stomach, caecum and colon (Figure 6). Treatment with either caspofungin or DHMB promoted C. albicans elimination from the mouse gut. For the gut microbiota, high numbers of E. coli and E. faecalis populations were observed following DSS treatment. Overgrowth of these two populations occurred regardless of C. albicans colonisation (Figure 7). DHMB treatment significantly maintained low levels of E. coli and E. faecalis populations while caspofungin treatment failed to reduce them. In terms of anaerobic bacteria, the number of L. johnsonii colonies was significantly reduced in both DSS and C. albicans mice, while DHMB treatment significantly restored L. johnsonii population in colitic mice challenged with C. albicans. In contrast, L. reuteri population showed unpredictable changes (Figure 7). The expression levels of pro- inflammatory cytokine IL-1β and anti-inflammatory cytokine IL-10 were assessed in the colons (Figure 8A,B). Expression of IL-1β was significantly lower in the colons of CaD mice treated with DHMB than in CaD or DSS mice (Figure 8A, 8B). In contrast, expression of IL-10 was significantly higher in colons of CaD mice challenged with DHMB when compared to that of CaD or DSS mice. Additionally, the expression levels of TLR-4 and TLR-8 were determined. TLR-4 and TLR-8

Int. J. Mol. Sci. 2019, 20, 321 9 of 15 Int. J. Mol. Sci. 2019, 20, 321 9 of 15 expression increased significantly in response to both C. albicans overgrowth and colitis while expression increased significantly in response to both C. albicans overgrowth and colitis while treatmentInt. J. Mol. Sci. with2019 DHMB, 20, 321 decreased the expression of these receptors in the colons (Figure 8A,B). 8 of 14 treatment with DHMB decreased the expression of these receptors in the colons (Figure 8A,B).

Figure 6. Effect of DHMB on C. albicans elimination from the gut. (A–D) Number of C. albicans colonies Figure 6. Effect of DHMB on C. albicans elimination from the gut. (A–D) Number of C. albicans colonies Figurerecovered 6. Effect from of the DHMB stools, on stomach, C. albicans cecum elimination and colo fromn. Data the gut. are (theA– Dmean) Number ± SD of C.10 albicans mice per colonies group recovered from the stools, stomach, cecum and colon. Data are the mean ± SD of 10 mice per group recoveredfrom two independentfrom the stools, experiments stomach, (cecump < 0.001). and colon. Data are the mean ± SD of 10 mice per group fromfrom two two independent independent experiments experiments ( (pp << 0.001). 0.001).

FigureFigure 7. 7. Determination ofof viableviable faecalfaecal aerobic aerobic and and anaerobic anaerobic bacteria bacteria in micein mice with with colitis colitis treated treated with FigurewithDHMB. DHMB. 7. For Determination all For experiments, all experiments, of viable stool bacteria stoolfaecal bacteria aerobic were collected were and collectedanae fromrobic mice from bacteria on mice Day in 0on beforemice Day with C.0 before albicans colitis C.challenge albicanstreated withchallengeand DHMB. DSS treatment.and For DSS all treatment. experiments, (A–D) Number (A– stoolD) ofNumber bacteriaE. coli, E.of were faecalisE. coli collected, ,E.L. faecalis johnsonii from, L. miceand johnsoniiL. on reuteri Day and 0colonies L.before reuteri C. recovered coloniesalbicans challengerecoveredfrom stools. and from Data DSS stools. are treatment. the Data mean (areA±– DSDthe) Number of mean 10 mice ±of perSDE. coli groupof , 10E. fromfaecalismice twoper, L. independentjohnsoniigroup from and experiments. twoL. reuteri independent colonies recoveredexperiments. from stools. Data are the mean ± SD of 10 mice per group from two independent experiments.

Int. J. Mol. Sci. 2019, 20, 321 9 of 14 Int. J. Mol. Sci. 2019, 20, 321 10 of 15

Figure 8. Cytokine and receptor expression after DHMB treatment. (A–D) Relative expression levels of FigureIL-1b, IL-10,8. Cytokine TLR-2 and and receptor TLR-8 mRNA expression in mouse after colons.DHMB Datatreatment are the. (A mean–D) Relative± SD of 10expression mice per levels group offrom IL-1b, two IL-10, independent TLR-2 and experiments. TLR-8 mRNA in mouse colons. Data are the mean ± SD of 10 mice per group from two independent experiments. 3. Discussion 3. Discussion The resistance of opportunistic yeast pathogens to antifungal drugs has increased significantly overThe the resistance past decade of andopportunistic this led us yeast to screen pathogens 55 molecules to antifungal from our drugs chemical has increased libraryfor significantly antifungal overactivity. the past DHMB decade was foundand this to beled a us promising to screen molecule 55 molecules in terms from ofMIC our valuechemical and library solubility. for Inantifungal addition, activity.DHMB wasDHMB not was cytotoxic foundin to vitro be aagainst promising a human molecule embryonic in terms kidney of MIC cell value line and (HEK293) solubility. at high In addition,concentrations DHMB (data was notnot shown).cytotoxic in vitro against a human embryonic kidney cell line (HEK293) at high concentrationsDHMB is often (data used not as a shown). key intermediate in the synthesis of different natural compounds such as theDHMB antibacterial is often agents used as (± a)-isoperbergin key intermediate and in perbergin the synthesis [14], the of different antineoplastic natural agent compounds combretastatin such asA-1 the [15 ],antibacterial the allergy-inducing agents benzofuranoquinone(±)-isoperbergin and acamelin perbergin [16], natural[14], the coumarins antineoplastic such as fraxetinagent combretastatinand fraxidin [ 17A-1], or[15], the the photoreactive allergy-inducing compound benzofuranoquinone pimpinellin [ 18acamelin], but the [16], role natural of this coumarins molecule suchalone as in fraxetin inflammation and fraxidin and fungal [17], eliminationor the photoreactive has not yet compound been studied. pimpinellin An hydroxy-deleted [18], but the role related of thiscompound, molecule 2-hydroxy-4-methoxybenzaldehyde alone in inflammation and fungal(HMB), elimination the principal has not componentyet been studied. of root An bark hydroxy- essential deletedoil of Periploca related sepiumcompound,Bunge, 2-hydroxy-4-methoxybenzaldehyde which is a woody climbing vine [19 ],(HMB), exhibits the some principal interesting component biological of rootactivities. bark essential The dried oil of root Periploca bark of sepium this plant Bunge, is traditionallywhich is a woody used climbi in theng treatment vine [19], of exhibits autoimmune some interestingdiseases, in biological particular activities. rheumatoid The dried arthritis root [20 bark]. It of also this appears plant is to traditionally have insecticidal used in activity the treatment against ofseveral autoimmune insect species diseases, [21 ].in particular rheumatoid arthritis [20]. It also appears to have insecticidal activityIn against the present several study, insect DHMB species was [21]. highly effective against clinically isolated fluconazole- or caspofungin-resistantIn the present study,C. albicans DHMBstrains was highly indicating effectiv thate thisagainst compound clinically could isolated potentially fluconazole- be used or to caspofungin-resistantfight drug-resistant fungi. C. albicans Some strains antifungal indicating drugs canthat inducethis compound changes incould the cellpotentially wall structure be used after to fightC. albicans drug-resistantchallenge. fungi. In contrast Some toantifungal caspofungin, drugs which can induce induces changes important in the cell cell wall wall changes structure in terms after of C.chitin albicans levels, challenge. DHMB In did contrast not induce to caspofungin, any important which changes induces in the important cell wall. cell DHMB wall waschanges also in involved terms ofin chitin the inhibition levels, DHMB of the melanin did not production. induce any It hasimportant been shown changes that in melanisation the cell wall. is a virulenceDHMB was factor also in involvedC. albicans inwhile the inhibition inhibition of melaninthe melanin biosynthesis production. by anti-melanin It has beenantibodies shown that leads melanisation to a decrease is ina virulencefungal virulence factor in [22 C.,23 albicans]. while inhibition of melanin biosynthesis by anti-melanin antibodies leads Into experimentala decrease in animals,fungal virulence we observed [22,23]. that the DHMB compound that we administered to animals at highIn experimental doses was not animals, cytotoxic we and observed was well-tolerated. that the DHMB The compound animals were that still we aliveadministered and healthy to animalstwo weeks at high after doses the end was of not the cytotoxic experiment. and was Furthermore, well-tolerated. the efficiency The animals of DHMB were still was alive assessed and healthyin vivo intwo the weeks DSS colitisafter the model. end of This the compound experiment. reduced Furthermore, the clinical the andefficiency histological of DHMB scores was for assessed in vivo in the DSS colitis model. This compound reduced the clinical and histological scores

Int. J. Mol. Sci. 2019, 20, 321 10 of 14 inflammation and promoted elimination of C. albicans from the gut. This finding is supported by a decrease in aerobic bacteria while anaerobic bacterial populations were re-established in mice treated with DHMB indicating that DHMB was not only able to reduce C. albicans colonisation but also, unexpectedly, to diminish intestinal inflammation. It has been shown that neutrophils and macrophages are recruited to the damaged colonic mucosa during DSS-induced colitis [24]. IL-1β expression, which is predominantly produced by these leukocytes, reduced in mice treated with DHMB. Recently, we observed after colitis development and C. glabrata challenge a high production of IL-6 and IL-1β, which are highly important in the recruitment of neutrophils and macrophages into the inflammatory site [9]. It has been shown that melanin concentrate hormone is highly expressed in the colonic mucosa of colitic mice and in patients with IBD suggesting that inhibition of melanin expression by DHMB can potentially reduce the pathogenesis of intestinal inflammation [25]. In conclusion, DHMB is a small organic molecule and any modification or substitution on the aromatic ring led to a strong decrease in the antifungal activity of the molecule, indicating that each group on the ring plays a specific and crucial role in its activity. This compound had antifungal properties against C. albicans, was safe and well-tolerated in different culture cell types and animals, and was effective at eliminating drug-resistant fungi, as well as had an anti-tyrosinase effect through the inhibition of the melanisation process in C. albicans. In DSS induced colitis model, DHMB had antifungal properties through the elimination of C. albicans from the gut and anti-inflammatory activity by re-establishing the anaerobic bacteria. This finding was also supported by a decrease of pro-inflammatory cytokine IL-1β in mice treaded with DHMB. Therefore, this aromatic small molecule holds great potential as an antifungal agent and anti-inflammatory properties.

4. Materials and Methods

4.1. C. albicans Strains and Growth Conditions The C. albicans strains used in the current study are shown in Table2. Fungal culture was carried out in YPD medium (yeast extract 1%, peptone 1%, dextrose 1%), on a rotary shaker for 18 h at 37 ◦C. The fungal culture obtained was then centrifuged at 2500 rpm for 5 min and washed twice in PBS (phosphate buffered saline).

Table 2. Antifungal activity of DHMB vs. caspofungin and ﬂuconazole.

MIC MIC MIC Strains Description Caspofungin Fluconazole DHMB Ref. (µg/mL) (µg/mL) (µg/mL) C. albicans ATCC 90028 Wild-type 0.03 0.5 8 This study C. albicans strain CAI4 Bioluminescent C. albicans 0.03 0.5 8 [26] (ura3::imm434/ura3::imm434) C. albicans 14314c4497 Anal, fluconazole resistant 0.06 128 80 This study C. albicans 14316c1746 Bronchoalveolar lavage, fluconazole resistant 0.03 128 80 This study C. albicans 92535989 Tracheal secretion, fluconazole resistant 0.06 64 80 This study C. albicans 14294c5335 Stools, fluconazole resistant 0.06 5 80 This study C. albicans 17292c3367 Venous catheter, caspofungin resistant 8 0.5 80 This study C. albicans 15343c3523 Blood, caspofungin resistant 2 0.5 80 This study C. albicans 17287c305 Blood, caspofungin resistant 8 0.5 80 This study C. albicans 15351c6859 Venous catheter, caspofungin resistant 4 1 80 This study

4.2. Antifungal Compounds The molecule 2,3-dihydroxy-4-methoxybenzaldehyde (DHMB) was synthesised and provided by Hautes Etudes d’Ingénieur (HEI), Lille, France. DHMB was used at a ﬁnal concentration of 100 µg/mL, diluted in PBS during the various in vitro experiments. Commercially available caspofungin (Merck, Semoy, France) and ﬂuconazole (Fresenius, Sèvres, France) were used as positive controls. Int. J. Mol. Sci. 2019, 20, 321 11 of 14

4.3. Fungal Viability Assays Minimum inhibitory concentrations (MICs) were measured using the broth microdilution method following Clinical Laboratory Standards Institute (CLSI) procedures. After drug inoculation, the plates were incubated at 37 ◦C for 24 h and MICs were measured as the lowest concentration of drug below growth levels [27]. For antifungal assays on C. albicans ATCC 90028, this screening was carried out by CO-ADD (Community for Antimicrobial Drug Discovery) and the University of Queensland (Santa Lucia, Australia). C. albicans ATCC 90028 was cultured for 3 days on YPD agar at 30 ◦C. A yeast suspension of 1 × 106 to 5 × 106 CFU/mL was prepared and the suspension was subsequently diluted and added to each well of the compound-containing plates giving a final cell density of fungi of 2.5 × 103 CFU/mL in a total volume of 50 µL. All plates were covered and incubated at 35 ◦C for 36 h without shaking. Growth inhibition of C. albicans ATCC 90028 was determined by measuring absorbance at 630 nm (OD630), after the addition of resazurin (0.001% final concentration) and incubation at 35 ◦C for 2 h. The absorbance was measured using a Biotek Multiflo Synergy HTX plate reader. The percentage growth inhibition was calculated for each well, using the negative control (media only) and positive control (fungi without inhibitor) on the same plate. The MIC was determined as the lowest concentration at which growth was fully inhibited, defined by an inhibition of ≥80% for C. albicans (total inhibition in the case of DHMB at 8 µg/mL concentration). For the viability assays, the bioluminescent C. albicans strain was suspended in PBS at a volume of 106 cells/well (96-well black plates, Chimney well). DHMB, caspofungin or fluconazole were then added at their final MIC concentrations (Table1). Coelenterazine was then added to the wells at a concentration of 2 µM. Bioluminescence kinetics were then measured (at 0, 30 and 60 min) and analysed with a FLUOstar Omega Fluorometer (BMG Labtech, Champigny sur Marne, France). The positive control consisted of C. albicans strain alone. The kinetics were also measured with the Xenogen device. For fungal CFU/mL counting, serial dilutions were performed by incubation of 105 C. albicans cells for 1 h with antifungal at the MIC concentration and an aliquot of 100 µL of each dilution was plated on Sabouraud dextrose agar and incubated for 48 h. For the melanin production, C. albicans yeast cells at 2 × 106 cells/mL were incubated in a minimal medium (15 mM glucose, 10 mM MgSO4, 29.4 mM KH2PO4, 13 mM glycine, 3 mM vitamin B1, pH 5.5) with or without 5 mM L-3,4-dihydroxyphenylalanine (DOPA, Sigma, St. Quentin Fallavier, France) at 37 ◦C in the dark. After 3 days, C. albicans cells grown with DOPA were treated with either DHMB (8 µg/mL) or caspofungin (0.0128 µg/mL). After one day of antifungal incubation, the production of melanin was examined using a Zeiss Axioplan microscope (Axioplan2, Jena, Germany).

4.4. Flow Cytometry and Confocal Microscopy C. albicans was suspended in PBS at a volume of 106 cells/well. DHMB, caspofungin or fluconazole were then added to each well at a final concentration of 100 µM. The expression of chitin or α-Mans was assessed using wheat germ agglutinin-fluorescein isothiocyanate and concanavalin A-rhodamine. The obtained data were analysed using Kaluza software. In parallel, a negative control without addition of lectin marker was prepared under the same conditions as those described previously for the samples. To examine the C. albicans cell wall by confocal microscopy after antifungal treatment, C. albicans cells were stained with wheat germ agglutinin-fluorescein isothiocyanate, concanavalin A-rhodamine and DAPI. Specific slides (well 6.7 mm; Thermo Scientific, Montigny le Bretonne, France) were used in this experiment and the coverslips were examined by confocal microscopy (Zeiss LSM710, Jena, Germany).

4.5. Animals The animals used were wild female C57BL/6 mice aged 3–4 months, certiﬁed free from infection and purchased from Janvier Laboratories, Le Genest-Saint-Isle, France. The mice were housed at the pet store of the Faculty of Medicine, Lille, France. The temperature of the room was maintained at 21 ◦C and the mice had free access to water and food with exposure to light 12 h/day. Water bottles Int. J. Mol. Sci. 2019, 20, 321 12 of 14 and food were daily examined in each cage before the evaluation of the clinical score for each tagged mouse. The studies were carried out in accordance with the decrees relating to the ethics of animal experimentation (Decree 86/609/EC, 8/2/2016).

4.6. C. albicans Challenge and Induction of Colitis Each mouse was administered with 300 µL of PBS containing 107 live C. albicans cells by oral gavage on Day 1. For antifungal treatment, mice were injected intraperitoneally for 5 days with 300 µL containing 10 mg/kg/day of DHMB or caspofungin. The DSS model is a reflection of how intestinal inflammation can promote C. albicans overgrowth in patients with CD. Mice received 2% DSS (M.W. 36−50 kDa; MP Biomedicals, LLC, Eschwege, Germany) in drinking water from Day 1 to Day 14 to promote colitis development and C. albicans intestinal colonisation [28]. The presence of C. albicans in the gut was assessed daily by measuring the number of CFUs in stools (approximately 0.1 g/sample). Stools were collected every 2 days from each tagged mouse. Faecal samples were suspended in 1 mL PBS, homogenised with a glass tissue homogeniser and plated onto Candi-Select medium (Bio-Rad Laboratories, Marnes la Coquette, France) [29]. The presence of C. albicans was assessed in the stomach, caecum and colon. The tissues were cut longitudinally and washed several times in PBS to avoid surface contamination from organisms present in the lumen. Serial dilutions of gut homogenates were prepared. The results were noted as C. albicans CFU/mg of tissue. For the isolation of aerobic bacteria, the faecal samples were plated onto MacConkey agar (Sigma-Aldrich, Saint Louis, MO, USA) and bile esculin azide agar (BEA, HongKong, China; Sigma-Aldrich, Saint Louis, MO, USA) plates. Serial dilutions of these samples were performed. For the isolation of anaerobic bacteria, Columbia agar (Sigma-Aldrich, Saint Louis, MO, USA) were used. MALDI-TOF MS (Microflex-Bruker Daltonics) was used to identify the bacteria [9].

4.7. Determination of Clinical and Histological Scores of Inflammation The clinical score was evaluated daily in each tagged mouse based on the presence of blood in the stools, rectal bleeding and stool consistency [5]. These three clinical markers of inflammation were recorded to give a score between 0 (healthy mouse) and 12 (maximum inflammation of the colon reflecting the severity of the inflammation). Colon samples were fixed in 4% paraformaldehyde-acid and embedded in paraffin for histological analysis. Histological scores were assessed by two independent, blinded investigators who observed two sections per mouse at magnifications of X10 and X100. The histological score was analysed according to two sub-scores: (i) a score for the presence of inflammatory cells; and (ii) a score for epithelial damage. The histological score ranged from 0 (no changes) to 6 (extensive cell infiltration and tissue damage) [5].

4.8. Real-Time mRNA Quantification of Pro-Inflammatory Cytokines and Innate Immune Receptors RNA extraction was performed with a NucleoSpin RNA® kit (Macherey-Nagel, Hoerdt, France). RNA was quantified by spectrophotometry (Nanodrop; Nyxor Biotech, Paris, France). cDNA synthesis was performed according to the High Capacity DNA Reverse Transcription (RT) protocol, using Master Mix (Applied Biosystems, Foster, CA, USA). Fast SYBR green (Applied Biosystems) was used to amplify the cDNA by PCR using the one-step system (Applied Biosystems). SYBR green dye intensity was assessed using one-step software. All results were normalised to the reference gene, POLR2A.

5. Statistical Analysis All data are presented as the mean ± standard deviation (SD) of individual experimental groups. Statistical analyses were performed using the Mann–Whitney U test to compare pairs of groups. Differences were considered signiﬁcant when the p value was as follows: p < 0.05; p < 0.01; p < 0.001. Prism 4.0 from GraphPad and XLSTAT were used for the statistical analyses. Int. J. Mol. Sci. 2019, 20, 321 13 of 14

Author Contributions: C.B., M.B., R.C., K.L., and S.J. performed the experiments. C.B., M.B., R.C., and S.J. analysed the data. C.B., M.B., R.C., K.L., A.G., B.S. and S.J. interpreted the results of the experiments. M.B., A.G., and B.S. contributed reagents/materials/analysis tools. S.J. designed the experiments and drafted the manuscript. Funding: This work was partially funded by Agence Nationale de la Recherche (ANR) in the setting the project “InnateFun”, promotional reference ANR-16-IFEC-0003-05, in the “Infect-ERA” program. Acknowledgments: The authors thank Nadine François and Antonino Bongiovanni for their excellent technical assistance. The authors would like to thank the Community for Antimicrobial Drug Discovery (CO-ADD), funded by the Welcome Trust (UK), and the University of Queensland (Australia) for performing the DHMB antimicrobial screening. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Baumgart, D.C.; Sandborn, W.J. Inflammatory bowel disease: Clinical aspects and established and evolving therapies. Lancet 2007, 369, 1641–1657. [CrossRef] 2. Oyri, S.F.; Muzes, G.; Sipos, F. Dysbiotic gut microbiome: A key element of Crohn’s disease. Comp. Immunol. Microbiol. Infect. Dis. 2015, 43, 36–49. [CrossRef][PubMed] 3. Gevers, D.; Kugathasan, S.; Denson, L.A.; Vazquez-Baeza, Y.; Van Treuren, W.; Ren, B.; Schwager, E.; Knights, D.; Song, S.J.; Yassour, M.; et al. The treatment-naive microbiome in new-onset Crohn’s disease. Cell Host Microbe 2014, 15, 382–392. [CrossRef][PubMed] 4. Darfeuille-Michaud, A.; Neut, C.; Barnich, N.; Lederman, E.; Di Martino, P.; Desreumaux, P.; Gambiez, L.; Joly, B.; Cortot, A.; Colombel, J.F. Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn’s disease. Gastroenterology 1998, 115, 1405–1413. [CrossRef] 5. Jawhara, S.; Thuru, X.; Standaert-Vitse, A.; Jouault, T.; Mordon, S.; Sendid, B.; Desreumaux, P.; Poulain, D. Colonization of mice by Candida albicans is promoted by chemically induced colitis and augments inflammatory responses through galectin-3. J. Infect. Dis. 2008, 197, 972–980. [CrossRef][PubMed] 6. Jawhara, S.; Poulain, D. Saccharomyces boulardii decreases inflammation and intestinal colonization by Candida albicans in a mouse model of chemically-induced colitis. Med. Mycol. 2007, 45, 691–700. [CrossRef] [PubMed] 7. Standaert-Vitse, A.; Sendid, B.; Joossens, M.; Francois, N.; Vandewalle-El Khoury, P.; Branche, J.; Van Kruiningen, H.; Jouault, T.; Rutgeerts, P.; Gower-Rousseau, C.; et al. Candida albicans colonization and ASCA in familial Crohn’s disease. Am. J. Gastroenterol. 2009, 104, 1745–1753. [CrossRef] 8. Poulain, D.; Sendid, B.; Standaert-Vitse, A.; Fradin, C.; Jouault, T.; Jawhara, S.; Colombel, J.F. Yeasts: Neglected pathogens. Dig. Dis. 2009, 27 (Suppl. 1), 104–110. [CrossRef] 9. Charlet, R.; Pruvost, Y.; Tumba, G.; Istel, F.; Poulain, D.; Kuchler, K.; Sendid, B.; Jawhara, S. Remodeling of the Candida glabrata cell wall in the gastrointestinal tract affects the gut microbiota and the immune response. Sci. Rep. 2018, 8, 3316. [CrossRef] 10. Ardizzone, S.; Cassinotti, A.; Manes, G.; Porro, G.B. Immunomodulators for all patients with inflammatory bowel disease? Ther. Adv. Gastroenterol. 2010, 3, 31–42. [CrossRef] 11. Campieri, M. New steroids and new salicylates in inflammatory bowel disease: A critical appraisal. Gut 2002, 50 (Suppl. 3), III43–III46. [CrossRef][PubMed] 12. Wiederhold, N.P. Antifungal resistance: Current trends and future strategies to combat. Infect. Drug Resist. 2017, 10, 249–259. [CrossRef][PubMed] 13. Ostrosky-Zeichner, L.; Casadevall, A.; Galgiani, J.N.; Odds, F.C.; Rex, J.H. An insight into the antifungal pipeline: Selected new molecules and beyond. Nat. Rev. Drug Discov. 2010, 9, 719–727. [CrossRef][PubMed] 14. Almabruk, K.H.; Chang, J.H.; Mahmud, T. Total Synthesis of (+/−)-Isoperbergins and Correction of the Chemical Structure of Perbergin. J. Nat. Prod. 2016, 79, 2391–2396. [CrossRef][PubMed] 15. Pettit, G.R.; Singh, S.B.; Niven, M.L.; Hamel, E.; Schmidt, J.M. Isolation, structure, and synthesis of combretastatins A-1 and B-1, potent new inhibitors of microtubule assembly, derived from Combretum caffrum. J. Nat. Prod. 1987, 50, 119–131. [CrossRef] 16. McKittrick, B.A.; Stevenson, R. A simple synthesis of the natural benzofuranoquinone, acamelin. J. Chem. Soc. Perkin Trans. 1 1983, 10, 2423–2424. [CrossRef] 17. Spath, E.; Dobrovolny, E. Natural coumarins. XLII. Synthesis of fraxetin, fraxidin and isofraxidin. Berichte der Deutschen Chemischen Gesellschaft 1938, 71B, 1831–1836. [CrossRef] Int. J. Mol. Sci. 2019, 20, 321 14 of 14

18. Cervi, A.; Aillard, P.; Hazeri, N.; Petit, L.; Chai, C.L.; Willis, A.C.; Banwell, M.G. Total syntheses of the coumarin-containing natural products pimpinellin and fraxetin using Au(I)-catalyzed intramolecular hydroarylation (IMHA) chemistry. J. Org. Chem. 2013, 78, 9876–9882. [CrossRef] 19. Wang, L.; Yin, Z.Q.; Zhang, L.H.; Ye, W.C.; Zhang, X.Q.; Shen, W.B.; Zhao, S.X. Chemical constituents from root barks of Periploca sepium. China J. Chin. Mater. Med. 2007, 32, 1300–1302. 20. Tokiwa, T.; Harada, K.; Matsumura, T.; Tukiyama, T. Oriental medicinal herb, Periploca sepium, extract inhibits growth and IL-6 production of human synovial fibroblast-like cells. Biol. Pharm. Bull. 2004, 27, 1691–1693. [CrossRef] 21. Chu, S.S.; Jiang, G.H.; Liu, W.L.; Liu, Z.L. Insecticidal activity of the root bark essential oil of Periploca sepium Bunge and its main component. Nat. Prod. Res. 2012, 26, 926–932. [CrossRef][PubMed] 22. Morris-Jones, R.; Gomez, B.L.; Diez, S.; Uran, M.; Morris-Jones, S.D.; Casadevall, A.; Nosanchuk, J.D.; Hamilton, A.J. Synthesis of melanin pigment by Candida albicans in vitro and during infection. Infect. Immun. 2005, 73, 6147–6150. [CrossRef][PubMed] 23. Eisenman, H.C.; Duong, R.; Chan, H.; Tsue, R.; McClelland, E.E. Reduced virulence of melanized Cryptococcus neoformans in Galleria mellonella. Virulence 2014, 5, 611–618. [CrossRef][PubMed] 24. Qualls, J.E.; Kaplan, A.M.; van Rooijen, N.; Cohen, D.A. Suppression of experimental colitis by intestinal mononuclear phagocytes. J. Leukoc. Biol. 2006, 80, 802–815. [CrossRef] 25. Kokkotou, E.; Moss, A.C.; Torres, D.; Karagiannides, I.; Cheifetz, A.; Liu, S.; O’Brien, M.; Maratos-Flier, E.; Pothoulakis, C. Melanin-concentrating hormone as a mediator of intestinal inflammation. Proc. Natl. Acad. Sci. USA 2008, 105, 10613–10618. [CrossRef] 26. Doyle, T.C.; Nawotka, K.A.; Kawahara, C.B.; Francis, K.P.; Contag, P.R. Visualizing fungal infections in living mice using bioluminescent pathogenic Candida albicans strains transformed with the firefly luciferase gene. Microb. Pathog. 2006, 40, 82–90. [CrossRef][PubMed] 27. Bustamante, B.; Martins, M.A.; Bonfietti, L.X.; Szeszs, M.W.; Jacobs, J.; Garcia, C.; Melhem, M.S. Species distribution and antifungal susceptibility profile of Candida isolates from bloodstream infections in Lima, Peru. J. Med. Microbiol. 2014, 63 Pt 6, 855–860. [CrossRef] 28. Choteau, L.; Parny, M.; Francois, N.; Bertin, B.; Fumery, M.; Dubuquoy, L.; Takahashi, K.; Colombel, J.F.; Jouault, T.; Poulain, D.; et al. Role of mannose-binding lectin in intestinal homeostasis and fungal elimination. Mucosal Immunol. 2016, 9, 767–776. [CrossRef] 29. Sendid, B.; Francois, N.; Standaert, A.; Dehecq, E.; Zerimech, F.; Camus, D.; Poulain, D. Prospective evaluation of the new chromogenic medium CandiSelect 4 for differentiation and presumptive identification of the major pathogenic Candida species. J. Med. Microbiol. 2007, 56 Pt 4, 495–499. [CrossRef]

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